Devices and Methods for White Blood Cell Analyses

ABSTRACT

Among other things, the present invention is related to devices and methods of performing biological and chemical assays, such as but not limited to assay related to analysis of white blood cells.

FIELD

Among other things, the present invention is related to devices andmethods of performing biological and chemical assays, such as but notlimited to assay related to analysis of white blood cells.

BACKGROUND

In biological and chemical assays (e.g. diagnostic testing), it is oftennecessary to measure and/or detect analytes of a sample or a part of thesample, quickly and simply. The current invention provides devices andmethods for achieving these goals.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way. In some Figures,the drawings are in scale. In the figures that present experimental datapoints, the lines that connect the data points are for guiding a viewingof the data only and have no other means.

FIG. 1 shows an embodiment of a QMAX (Q: quantification; M: magnifying;A: adding reagents; X: acceleration; also known as compressed regulatedopen flow (CROF)) device, which comprises a first plate and a secondplate. Panel (A) shows the perspective view of the plates in an openconfiguration when the plates are separated apart; panel (B) shows theperspective view and a sectional view of depositing a sample on thefirst plate at the open configuration; panel (C) the perspective viewand a sectional view of the QMAX device in a closed configuration.

FIG. 2 illustrates white blood cell (WBC) counting accuracy vs. field ofview (FoV) vs. QMAX gap (thickness of sample layer). Panel (A) showsplots of WBC counting accuracy vs. QMAX gap size with effective FoV of 4mm², 16 mm², 36 mm², 64 mm², and 100 mm²; panel (B) shows plots of WBCcounting accuracy FoV with QMAX gap size of 2 um, 3 um, 5 um, 6.2 um, 10um and 30 um.

FIG. 3 shows a schematic exploded view of an optical adaptor device forattaching the QMAX device to a mobile communication device.

FIG. 4 shows a schematic sectional view with details of a system thatcan be used to test a sample in fluorescent illumination mode, andparticularly of the optical adapter.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description illustrates some embodiments of theinvention by way of example and not by way of limitation. The sectionheadings and any subtitles used herein are for organizational purposesonly and are not to be construed as limiting the subject matterdescribed in any way. The contents under a section heading and/orsubtitle are not limited to the section heading and/or subtitle, butapply to the entire description of the present invention.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

Examples of QMAX Device with Hinges (QMAX Card)

FIG. 1 shows an embodiment of a generic QMAX (Q: quantification; M:magnifying; A: adding reagents; X: acceleration; also known ascompressed regulated open flow (CROF)) device. The generic QMAX devicecomprises a first plate 10 and a second plate 2. In particular, panel(A) shows the perspective view of a first plate 10 and a second plate 20wherein the first plate has spacers. It should be noted, however, thatthe spacers can also be fixed on the second plate 20 (not shown) or onboth first plate 10 and second plate 20 (not shown). Panel (B) shows theperspective view and a sectional view of depositing a sample 90 on thefirst plate 10 at an open configuration. It should be noted, however,that the sample 90 also can also be deposited on the second plate 20(not shown), or on both the first plate 10 and the second plate 20 (notshown). Panel (C) illustrates (i) using the first plate 10 and secondplate 20 to spread the sample 90 (the sample flow between the innersurfaces of the plates) and reduce the sample thickness, and (ii) usingthe spacers and the plate to regulate the sample thickness at the closedconfiguration of the QMAX device. The inner surfaces of each plate haveone or a plurality of binding sites and or storage sites (not shown).

In some embodiments, the spacers 40 have a predetermined uniform heightand a predetermined uniform inter-spacer distance. In the closedconfiguration, as shown in panel (C) of FIG. 1, the spacing between theplates and the thus the thickness of the sample 90 is regulated by thespacers 40. In some embodiments, the uniform thickness of the sample 90is substantially similar to the uniform height of the spacers 40. Itshould be noted that although FIG. 1 shows the spacers 40 to be fixed onone of the plates, in some embodiments the spacers are not fixed. Forexample, in certain embodiments the spacers are mixed with the sample sothat when the sample is compressed into a thin layer, the spacers, whichis rigid beads or particles that have a uniform size, regulate thethickness of the sample layer.

QMAX Assay

In biological and chemical assaying (i.e. testing), a device and/or amethod that simplifies assaying operation or accelerates assaying speedis often of great value.

In the QMAX (Q: quantification; M: magnifying; A: adding reagents; X:acceleration; also known as compressed regulated open flow (CROF)) assayplatform, a QMAX card uses two plates to manipulate the shape of asample into a thin layer (e.g. by compressing) (as illustrated in FIG.1). In certain embodiments, the plate manipulation needs to change therelative position (termed: plate configuration) of the two platesseveral times by human hands or other external forces. There is a needto design the QMAX card to make the hand operation easy and fast.

In QMAX assays, one of the plate configurations is an openconfiguration, wherein the two plates are completely or partiallyseparated (the spacing between the plates is not controlled by spacers)and a sample can be deposited. Another configuration is a closedconfiguration, wherein at least part of the sample deposited in the openconfiguration is compressed by the two plates into a layer of highlyuniform thickness, the uniform thickness of the layer is confined by theinner surfaces of the plates and is regulated by the plates and thespacers.

In a QMAX assay operation, an operator needs to first make the twoplates to be in an open configuration ready for sample deposition, thendeposit a sample on one or both of the plates, and finally close theplates into a close position. In certain embodiments, the two plates ofa QMAX card are initially on top of each other and need to be separatedto get into an open configuration for sample deposition. When one of theplate is a thin plastic film (175 um thick PMA), such separation can bedifficult to perform by hand. The present invention intends to providethe devices and methods that make the operation of certain assays, suchas the QMAX card assay, easy and fast.

In some embodiments, the QMAX device comprises a hinge that connects thetwo or more plates, so that the plates can open and close in a similarfashion as a book.

In certain embodiments, the hinge is configured so that the hinge canself-maintain the angle between the plates after adjustment.

In certain embodiments, the hinge is configured so that the material ofthe hinge, which maintain the QMAX card in the closed configuration,such that the entire QMAX card can be slide in and slide out a card slotwithout causing accidental separation of the two plates.

Another aspect of the present invention is to provide opening mechanismssuch as but not limited to notches on plate edges or strips attached tothe plates, making is easier for a user to manipulate the positioning ofthe plates, such as but not limited to separating the plates of by hand.

Another aspect of the present invention is to provide a hinge that cancontrol the rotation of more than two plates.

The term “compressed open flow (COF)” refers to a method that changesthe shape of a flowable sample deposited on a plate by (i) placing otherplate on top of at least a part of the sample and (ii) then compressingthe sample between the two plates by pushing the two plates towards eachother; wherein the compression reduces a thickness of at least a part ofthe sample and makes the sample flow into open spaces between theplates. The term “compressed regulated open flow” or “CROF” (or“self-calibrated compressed open flow” or “SCOF” or “SCCOF”) (also knownas QMAX) refers to a particular type of COF, wherein the final thicknessof a part or entire sample after the compression is “regulated” byspacers, wherein the spacers are placed between the two plates. Here theCROF device is used interchangeably with the QMAX device.

The term “spacers” or “stoppers” refers to, unless stated otherwise, themechanical objects that set, when being placed between two plates, alimit on the minimum spacing between the two plates that can be reachedwhen compressing the two plates together. Namely, in the compressing,the spacers will stop the relative movement of the two plates to preventthe plate spacing becoming less than a preset (i.e. predetermined)value.

The term “a spacer has a predetermined height” and “spacers have apredetermined inter-spacer distance” means, respectively, that the valueof the spacer height and the inter spacer distance is known prior to aQMAX process. It is not predetermined, if the value of the spacer heightand the inter-spacer distance is not known prior to a QMAX process. Forexample, in the case that beads are sprayed on a plate as spacers, wherebeads are landed at random locations of the plate, the inter-spacerdistance is not predetermined. Another example of not predeterminedinter spacer distance is that the spacers moves during a QMAX processes.

The term “a spacer is fixed on its respective plate” in a QMAX processmeans that the spacer is attached to a location of a plate and theattachment to that location is maintained during a QMAX (i.e. thelocation of the spacer on respective plate does not change) process. Anexample of “a spacer is fixed with its respective plate” is that aspacer is monolithically made of one piece of material of the plate, andthe location of the spacer relative to the plate surface does not changeduring the QMAX process. An example of “a spacer is not fixed with itsrespective plate” is that a spacer is glued to a plate by an adhesive,but during a use of the plate, during the QMAX process, the adhesivecannot hold the spacer at its original location on the plate surface andthe spacer moves away from its original location on the plate surface.

The term “open configuration” of the two plates in a QMAX process meansa configuration in which the two plates are either partially orcompletely separated apart and the spacing between the plates is notregulated by the spacers

The term “closed configuration” of the two plates in a QMAX processmeans a configuration in which the plates are facing each other, thespacers and a relevant volume of the sample are between the plates, therelevant spacing between the plates, and thus the thickness of therelevant volume of the sample, is regulated by the plates and thespacers, wherein the relevant volume is at least a portion of an entirevolume of the sample.

The term “a sample thickness is regulated by the plate and the spacers”in a QMAX process means that for a give condition of the plates, thesample, the spacer, and the plate compressing method, the thickness ofat least a port of the sample at the closed configuration of the platescan be predetermined from the properties of the spacers and the plate.

The term “inner surface” or “sample surface” of a plate in a QMAX devicerefers to the surface of the plate that touches the sample, while theother surface (that does not touch the sample) of the plate is termed“outer surface”.

The term “height” or “thickness” of an object in a QMAX process refersto, unless specifically stated, the dimension of the object that is inthe direction normal to a surface of the plate. For example, spacerheight is the dimension of the spacer in the direction normal to asurface of the plate, and the spacer height and the spacer thicknessmeans the same thing.

The term “area” of an object in a QMAX process refers to, unlessspecifically stated, the area of the object that is parallel to asurface of the plate. For example, spacer area is the area of the spacerthat is parallel to a surface of the plate.

The term of QMAX device refers the device that perform a QMAX (e.g.CROF) process on a sample, and have or not have a hinge that connect thetwo plates.

The term “QMAX device with a hinge and “QMAX card” are interchangeable.

The term “angle self-maintain”, “angle self-maintaining”, or “rotationangle self-maintaining” refers to the property of the hinge, whichsubstantially maintains an angle between the two plates, after anexternal force that moves the plates from an initial angle into theangle is removed from the plates.

QMAX Device and Assay for Cell Counting

The QMAX device can be used to analyze fluid samples, such as but notlimited to biological fluid samples. In some embodiments, the QMAXdevice is used to analyze a blood sample. For example, in certainembodiments, the QMAX device is used to measure the amount of certainanalytes, e.g. counting of red blood cells (RBC), white blood cells(WBC), and/or subtypes of certain blood cells. In certain embodiments,the QMAX device can be used for the counting of WBC. In certainembodiments, staining reagents can be used to label the cells andstructures, such as but not be limited to RBC, WBC (including WBCsubtypes), and platelets.

As shown in FIG. 1, various parameters of the QMAX device can vary basedon specific tests. For example, in some embodiment, the spacer height isless than 0.2 um, 0.5 um, 0.8 um, 1 um, 1.2 um, 1.5 um, 1.8 um, 2 um, 3um, 4 um, 5 um, 6 um, 7 um, 8 um, 9 um, 10 um, 11 um, 12 um, 13 um, 14um, 15 um, 16 um, 17 um, 18 um, 19 um, 20 um, 25 um, 30 um, 35 um, 40um, 45 um, 50 um, 60 um, 70 um, 75 um, 80 um, 90 um, 100 um, 125 um, 150um, 175 um, 200 um, 250 um, 300 um, 350 um, 400 um, 450 um, 500 um, 600um, 700 um, 800 um, 900 um, 1 mm, 2 mm, 3 mm, 4 mm, 5mm, 10 mm, or in arange between any of the two values. In the closed configuration, theuniform thickness of the sample layer is substantially the same as thegap between the QMAX plates, which is substantially the same as thespacer height. Therefore, the descriptions to the spacer height alsoapply to the thickness of the sample layer and the QMAX gap, and viceversa.

In some embodiments of the QMAX assay, the sample is deposited to one orboth of the plates in the open configuration; then the plates arepressed into a closed configuration so that at least part of the samplecompressed into a layer of highly uniform thickness, which is stagnantto the plates and confined by the inner surfaces of the plates. In someembodiments, an analyte in the sample is measured. In certainembodiments, the analyte is a type of cells that can be counted. Forexample, in certain embodiments the sample is a blood sample and theanalyte is red blood cells; in certain embodiments the sample is a bloodsample and the analyte is white blood cells; in certain embodiments thesample is a blood sample and the analyte is white blood cell sub-types(including neutrophils, eosinophils, basophils, lymphocytes, andmonocytes).

In some embodiments, when the QMAX device is in the closedconfiguration, a camera can be used to capture images of the samplelayer. In certain embodiments, the camera can have a field of view(FoV), which is defined as the area of sample of which the image can becaptured by the camera. In certain embodiments, the camera is part of adevice, such as but not limited to a mobile device. In certainembodiments, the mobile device is a smart phone, a tablet computer, or alaptop computer. In some embodiments, the mobile device is a mobilecommunication device such as a smart phone. In certain embodiments, thecamera has one lens; in certain embodiments, the camera has two lensesthat are aligned parallel to each other.

In some embodiments, different spacer height (hence different samplethickness and QMAX gap) can affect the accuracy of the counting ofcertain cells, such as but not limited to white blood cells andsub-types of white blood cells. For example, for counting white bloodcells (WBC), spacer height and FoV can affect the accuracy andconsistency of the counting results. With an acceptable level ofconsistency, the direct counting results can be adjusted to reflect thereal number of cells, providing basis for diagnostics and healthguidance. In certain embodiments, one factor that needs to be consideredis the consistency of “miss count” rate, which is the deviation of theresults with a method being tested from the real number, which isusually established with a well-defined and well-accepted method. Itshould also be noted that the method herein disclosed can be applied tonot only WBC counting, but also other assays.

The device and method of the current invention can be used to (1) countthe white blood cells, (b) count the white blood cells sub-types(including neutrophils, eosinophils, basophils, lymphocytes, andmonocytes), and (3) differentiate white blood cells, wherein the devicefurther comprises spacers that regulate the spacing between the samplecontact areas when the plates are in a closed configuration.

In some embodiments, the average thickness of the layer of uniformthickness is in the range of 5.0 um to 8.5 um.

In some embodiments, the average thickness of the layer of uniformthickness is in the range of 7.5 um to 10.5 um.

In some embodiments, the average thickness of the layer of uniformthickness is in the range of 9.5 um to 12.5 um.

In some embodiments, the average thickness of the layer of uniformthickness is in the range of 9.5 um to 12.5 um.

In some embodiments, the average thickness of the layer of uniformthickness is in the range of 11.5 um to 13.5 um.

In some embodiments, the average thickness of the layer of uniformthickness is in the range of 12.5 um to 14.5 um.

In some embodiments, the average thickness of the layer of uniformthickness is in the range of 13.5 um to 16 um.

In some embodiments, the spacer height is in the range of 5.0 um to 8.5um.

In some embodiments, the spacer height is in the range of 7.5 um to 10.5um.

In some embodiments, the spacer height is in the range of 9.5 um to 12.5um.

In some embodiments, the spacer height is in the range of 9.5 um to 12.5um.

In some embodiments, the spacer height is in the range of 11.5 um to13.5 um.

In some embodiments, the spacer height is in the range of 12.5 um to14.5 um.

In some embodiments, the spacer height is in the range of 13.5 um to 16um.

In some embodiments, the field of view for counting and differentiatingWBCs is 0.1 mm², 10 mm², 50 mm², 100 mm² or a range between any two ofthe values.

In some embodiments, when the gap size of the QMAX device is about 10um, the FoV is larger than 36 mm², thereby the WBC counting anddifferentiation accuracy is less than 5%.

In some embodiments, when the gap size of device is 10 um, the FoV islarger than 16 mm², thereby the WBC counting and differentiationaccuracy is less than 10%.

In some embodiments, when the gap size of device is 10 um, the FoV islarger than 2 mm², thereby the WBC counting and differentiation accuracyis less than 20%.

In some embodiments, the field of view is in the range of 0.1 mm² to 10mm², the preferred gap size of device is in the range of 10 um to 30 um,30 um to 50 um, thereby the counting and differentiation accuracy isless than 10%.

In some embodiments, the field of view is in the range of 0.1 mm² to 10mm², the preferred gap size of device is in the range of 10 um to 30 um,thereby the counting and differentiation accuracy is less than 20%.

In some embodiments, the field of view is in the range of 10 mm² to 50mm², the preferred gap size of device is in the range of 5 um to 10 um,10 um to 30 um, thereby the counting and differentiation accuracy isless than 10%.

In some embodiments, the field of view is in the range of 10 mm² to 50mm², the preferred gap size of device is in the range of 2 um to 5 um, 5um to 10 um, 10 um to 30 um, thereby the counting and differentiationaccuracy is less than 20%.

In some embodiments, the field of view is in the range of 50 mm² to 100mm², preferred gap size of device is in the range of 2 um to 5 um, 5 umto 10 um, 10 um to 30 um, 30 um to 50 um thereby the counting anddifferentiation accuracy is less than 10%.

In some embodiments, the spacer has a height of preferred range of 2 umto 5 um, thereby the WBCs missing counting is less than 15%.

In some embodiments, the spacer has a height of preferred range of 2 umto 5 um, 5 um to 10 um, thereby the WBCs missing counting is less than30%.

In some embodiments, the spacer has a height of preferred range of 2 umto 5 um, 5 um to 10 um, 10 um to 30 um thereby the WBCs missing countingis less than 60%.

In some embodiments, the sample to camera lens distance is in the rangeof 2 mm to 5 mm.

In some embodiments, the sample to camera lens distance is in the rangeof 4 mm to 7 mm.

In some embodiments, the sample to camera lens distance is in the rangeof 6 mm to 9 mm.

In some embodiments, the sample to camera lens distance is in the rangeof 8 mm to 11 mm.

In some embodiments, the sample to camera lens distance is in the rangeof 10 mm to 13 mm.

In some embodiments, the sample to camera lens distance is in the rangeof 12 mm to 15 mm.

Examples of QMAX Device for Counting White Blood Cells

FIG. 2 illustrates white blood cell (WBC) counting accuracy vs. field ofview (FoV) vs. QMAX gap (thickness of sample layer). Undiluted blood wasdeposited on one or both of the plates of the QMAX device in the openconfiguration; the plates were pressed into a closed configuration sothat at least part of the sample was compressed into a layer of uniformthickness; a camera in a smart phone was used to capture images of thecompressed sample; the number of WBC was counted by analyzing theimages.

Panel (A) of FIG. 2 shows plots of WBC counting accuracy vs. QMAX gapsize with effective FoV of 4 mm², 16 mm², 36 mm², 64 mm², and 100 mm²;panel (B) shows plots of WBC counting accuracy FoV with QMAX gap size of2 um, 3 um, 5 um, 6.2 um, 10 um and 30 um. The results are alsosummarized in Table 1.

TABLE 1 WBC counting accuracy vs. Field of View vs. QMAX gap Field ofView QMAX gap size (um) (mm²) 2 3 5 6.2 10 30 4 61% 57% 39%  24%  15% 12%  16 33% 27% 15%  12%  8% 8% 36 20% 13% 8% 7% 4% 4% 64  7%  7% 6% 6%3% 2% 100  6%  6% 3% 5% 2% 3%

In this set of experiments, the first plate of the QMAX device is 1 mmthick PMMA with printed acridine orange dye, and the second plate isX-Plate with spacers having 30×40 um pillar size, 80 um inter spacingdistance, made on 175 um thick PMMA. 1 uL fresh blood without anyanticoagulant was used in the test and deposited on the first plate.Counting accuracy is defined as the counting number's standard deviationfor all the fields on card with a specific FoV. This counting accuracyrepresents the case when a field with FoV in the sample layer israndomly picked for measure, how accurate it represents the averagenumber of all the fields. Generally, WBC counting is more accurate withlarger field of view and larger QMAX gap. In essence, counting accuracyhere reflects the consistency of the method with specific gap size andfield of view.

Table 2 shows in certain case of using fluorescence measuring WBC, therelationship between WBC miss counting and correction factor vs. QMAXgap. Herein, miss counting rate is defined as the percentage differencebetween the back-calculated WBC concentration (from counting number,counting area, filling factor, gap size) and sample's real WBCconcentrations (measured by calibrated commercial hematology machine).

Correction factor=1/(1−Missing Counting Rate).

TABLE 2 WBC miss counting & correction factor vs. QMAX gap QMAX gap size(um) WBC miss counting WBC correction factor 2  0% 1 3  0% 1 5 10% 1.110 25% 1.3 30 50% 2.0

As shown in Table 2, the miss counting rate increases with the gap size(thus spacer height and sample thickness). Furthermore, additionalexperiments show that differentiated WBC (Granulocytes, Lymphocyte,Monocyte) counting has similar miss counting rate with WBC totalcounting. In addition, WBC miss counting rate is not influenced by fieldof view.

As shown in FIG. 2, panels (A) and (B), the counting accuracy, whichreflects the consistency of the counting at certain gap sizes and fieldof view, is higher with a larger gap size and a larger field of view,respectively. Therefore, in some embodiments, certain gap sizes (thusspacer heights) and/or field of view size can be chosen to obtain anacceptable level of consistency, and/or prevent high level of misscount.

With the correction factor, which is based on the miss counting rate,the counting result can be adjusted to provide a more accurate andconsistent number for medical and health purposes. In some embodiments,the final number equals the counting results multiplies the correctionfactor.

Table 3 shows the calculation of self-overlap rate of WBC cell vs. QMAXgap. In general, more WBCs are overlapped when the gap size is larger,especially larger than 30 um.

TABLE 3 QMAX gap size vs. WBC distance vs. Overlap rate CROF gap (um)Cell 2D Distance (um) Overlap Rate 2 320 0% 10 140 0% 30 80 1% 50 60 2%70 50 5% 100 45 9% 300 25 66% Exemplary Embodiments with a Gap of 8 to 12 um

The experiments (see e.g. FIGS. 2-3) show that for the measurement ofWBC in undiluted blood sample, with a given field of view provide by acamera (e.g. camera in a mobile phone), a spacer height of 5 to 15 umprovides more accurate results than spacer height of 2 um to 3 um. Insome embodiments, a QMAX device for WBC measurement has spacer height of5 to 15 um. In certain embodiments, the QMAX device has a spacer heightof 10 um, while a same of a similar sample thickness uniformity can beachieved. In some embodiments, such pillar heights have advantage forimaging and counting the white blood cells in an undiluted blood.

Exemplary Embodiments of Optical Adapter

In some embodiments, the QMAX device (e.g. in the form of a QMAX card)with sample can be inserted into an adaptor, which can be attached to adevice that comprises a camera and/or an illumination source. In certainembodiments, the device is a mobile communication device, such as butnot limited to a smart phone.

FIG. 3 shows a schematic exploded view of an optical adaptor device forattaching the QMAX device to a mobile communication device and formeasurement of an analyte in the sample. Here the optical adaptor device18 is in system 19, which comprises the mobile communication device(smart phone) 1.

Adaptor 18 comprises a holder case 2 fitting over the upper part ofsmartphone 1; an optical box 3 attached to case 2 including a receptacleslot 4, an optics chamber 3C, track 6 b and 6 t allowing lever 8 toslide in, and a rubber door 16 inserted into trench 4 s to coverreceptacle slot 4. An optics insert 7 is fitted into the top of opticschamber 3C with an exit aperture 7L and an entrance aperture 7C in italigning with light source 1L and camera 1C (referring to FIG. 4) insmartphone 1. A lens 11 is mounted in entrance aperture 7C in opticsinsert 7 and configured so that the sample in sample slide 5 insertedinto receptacle slot 4 is located within the working distance of thecamera 1C (referring to FIG. 4). Lens 11 serves to magnify the images ofthe sample captured by camera 1C (referring to FIG. 4). A long-passoptical filter 12 is mounted on top of lens 11 in entrance aperture 7C.A pair of right-angle mirrors 13 and 14 are mounted on the bottom ofoptics chamber 3C and configured so that mirror 13 and mirror 14 arealigned with light source 1L and camera 1C (referring to FIG. 4)respectively. Mirror 13 and mirror 14 whose operation as bright-fieldillumination optics in device 18 is described below in FIG. 4.

Lever 8 comprises two level bars: the upper-level bar comprises aband-pass optical filter 15 mounted in slot 8 a, and the lower-level barcomprises a light absorber 9 mounted on the horizontal plane 8 b and areflective mirror 10 mounted on the tilted plane 8 c. The optical filter15, light absorber 9 and mirror 10 whose operation as fluorescentillumination optics in device 18 is described in FIG. 4. The upper-levelbar of lever 8 slides along track 6 t in box 3 and lower-level bar 8 band 8 c slides along track 6 b in box 3. Lever 8 stops at two differentpositions in box 3 to switch between bright-field illumination opticsand fluorescent illumination optics. Lever 8 is fully inserted into box3 to switch device 18 to work with fluorescent illumination optics. Ballplunger 17 is mounted on the sidewall of track 6 t to stop lever 8 at apre-defined position when lever 8 being pulled outward from box 3 toswitch device 18 to work with bright-field illumination optics.

FIG. 4 shows a schematic sectional view with details of a system thatcan be used to test a sample in fluorescent illumination mode, andparticularly of the optical adaptor. This FIG. 4 illustrates thefunctionality of the elements that were described above with referenceto FIG. 3. Lever 8 (shown in FIG. 3) is fully inserted into device 18 sothat light absorber 9 and tilted mirror 10 are under the view of camera1C and light source 1L, and block the light path between light source 1Land the pair of mirrors of 13 and 14. And band-pass optical filter 15 isright under the light source 1L. Light source 1L emits light beam BF1away from smartphone 1. Optical filter 15 allows beam BF1 with specificwavelength range which matches the excitation wavelength of thefluorescent sample in sample slide 5 to go through. Part of beam BF1illuminates on the edge of transparent sample slide 5 and couples towaveguide beam BF3 travelling in sample slide 5 and illuminates thesample area under the lens 11. Part of beam BF1 illuminates on mirror10. Tilted mirror 10 deflects beam BF1 to beam BF2 and back-illuminatesthe sample area in sample slide 5 right under lens 11 in large obliqueangle. The remaining part of beam BF1 with large divergence angle (i.e.,beam BF4) illuminates on absorber 9 and get absorbed so that noreflected light of beam BF4 gets into the camera 1C in small incidenceangle. The light coming from the sample area under the lens 11 goesthrough the lens 11 and is filtered by long-pass filter 12 so that onlylight in a specify wavelength range that is emitted by the fluorescentsample in sample slide 5 gets into camera 1C to form an image.Smartphone 1 captures and processes the image to get some property ofthe sample. Rubber door 16 is inserted into device 18 to cover sampleslide 5 to prevent ambient light getting into device 18 to affect thetest.

In some embodiments, the adapter as described in FIGS. 3 and 4 can beused to measure a blood sample, e.g. undiluted whole blood sample. Incertain embodiments, the analyte can be WBC, which requires the lever 8to be inserted for optimal reading. In some embodiments, the adaptercomprises:

-   -   (a) an attachment member configured to attach the adapter to an        apparatus that comprises a light source and a camera;    -   (b) a card slot configured to accommodate a sample card, which        contains a liquid sample that is compressed into a layer of        uniform thickness, wherein when the sample card inserted into        the card slot, the sample is positioned under the view of the        camera and the light source;    -   (c) an optical filter configured to filter light from the light        source to form a first beam with a specific wavelength range,        wherein a part of the first beam illuminates on the edge of the        sample card and travels in the sample card to illuminate the        sample;    -   (d) a mirror configured to deflect part of the first beam to        form a second beam that back-illuminates the sample in an        oblique angle;    -   (e) an absorber configured to absorb a remaining part of the        first beam that has a divergence angle.

In some embodiments, the method to measure an analyte, such as but notlimited to WBC, in a liquid sample, can comprises:

(a) obtaining the liquid sample;

(b) compressing at least part of the sample into a layer of uniformthickness with a sample card,

(c) inserting the sample card into an adaptor device, which isconfigured to be attached to an apparatus that comprises a light sourceand a camera;

(d) illuminating the sample with light from the light source, wherein

-   -   i. the light is filtered by an optical filter of the adapter        device to form a first beam with a specific wavelength range,        part of the first beam illuminating on the edge of the sample        card and travels in the sample card to illuminate the sample;    -   ii. part of the first beam is deflected by a mirror of the        adapter device to form a second beam that back-illuminates the        sample in an oblique angle; and    -   iii. a remaining part of the first beam that has a divergence        angle is absorbed by an absorber of the adapter device.

In some embodiment, the method can further comprise:

(e) capturing images of the sample in the layer of uniform thicknesswith the camera;

(f) analyzing the images to enumerate the analyte in the images; and

(g) calculating the concentration of the analyte in the sample based onthe uniform thickness, a field of view of the camera, analyte number,and a predetermined correction factor;

wherein the field of view is the extent of the field in which the cameracaptures the images;

wherein the correction factor is determined by a miscount ratio, whichis dependent on the field of view, the uniform thickness, and propertiesof the analyte.

Exemplary Embodiments for WBC Measurement

For the device or method embodiments of the current invention, thedevice can further comprise, on one or both plates, multi reagent layersincluding anti-conglutination reagents, cell lysing reagents, cellstaining reagents, release time control material, and any combinationsthereof.

In some embodiments, each reagent layer coated on the plates has athickness of 10 nm, 100 nm, 200 nm, 500 nm, 1 um or in a range betweenany two of the values.

In some embodiments, the anti-conglutination agent comprisesethylenediaminetetraacetic acid (EDTA), EDTA disodium, K2EDTA, orK3EDTA, or any combinations thereof.

In some embodiments, the cell stain agent comprises Wright's stain(Eosin, methylene blue), Giemsa stain (Eosin, methylene blue, and AzureB), May-Grünwald stain, Leishman's stain (“Polychromed” methylene blue(i.e. demethylated into various azures) and eosin), Erythrosine B stain(Erythrosin B), and other fluorescence stain including but not limit toAcridine orange dye, 3,3-dihexyloxacarbocyanine (DiOC6), PropidiumIodide (PI), Fluorescein Isothiocyanate (FITC) and Basic Orange 21(BO21) dye, Ethidium Bromide, Brilliant Sulfaflavine and a StilbeneDisulfonic Acid derivative, Erythrosine B or trypan blue, Hoechst 33342,Trihydrochloride, Trihydrate, or DAPI (4′,6-Diamidino-2-Phenylindole,Dihydrochloride), or any combinations thereof.

In some embodiments, the cell lysing agent comprises ammonium chloride,sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), acetic acid,citric acid, or other acid and base, or any combinations thereof.

In some embodiments, the release time control material comprisesalbumin, carbomers, carboxymethyl cellulose, carrageenan, chitosan,dextrin, polyethylene glycol, polyvinylpyrrolidone, or polyvinylalcohol, or any combinations thereof.

In some embodiments of the method embodiments of the current invention,the RBCs, platelets, or both are lysed in the sample before thedetection and/or measurement of WBCs.

In some embodiments of the method embodiments of the current invention,the WBCs, platelets, or both are lysed in sample before the detection ofRBCs.

In some embodiments of the method embodiments of the current invention,the RBCs, WBCs, or both are lysed in sample before the detection ofPLTs.

Group of Other Examples of Present Invention

Further examples of inventive subject matter according to the presentdisclosure are described in the following enumerated paragraphs.

Correction Factor and Field of View

-   A1. A method for analyzing an analyte in a liquid sample,    comprising:

(a) obtaining the liquid sample;

(b) compressing at least part of the sample into a layer of uniformthickness,

(c) capturing images of the sample in the layer of uniform thicknesswith a camera, wherein the images show the analyte; and

(d) analyzing the images to enumerate the analyte in the images,

(e) calculating the concentration of the analyte in the sample based onthe uniform thickness, a field of view of the camera, the analyteenumeration, and a predetermined correction factor;

wherein the field of view is the extent of the field in which the cameracaptures the images;

wherein the correction factor is determined by a miscount ratio, whichis dependent on the field of view, the uniform thickness, and propertiesof the analyte.

Illumination for WBC

-   B1. An adapter device for analyzing an analyte in a liquid sample,    comprising:    -   (f) an attachment member configured to attach the adapter to an        apparatus that comprises a light source and a camera;    -   (g) a card slot configured to accommodate a sample card, which        contains a liquid sample that is compressed into a layer of        uniform thickness, wherein when the sample card inserted into        the card slot, the sample is positioned under the view of the        camera and the light source;    -   (h) an optical filter configured to filter light from the light        source to form a first beam with a specific wavelength range,        wherein a part of the first beam illuminates on the edge of the        sample card and travels in the sample card to illuminate the        sample;    -   (i) a mirror configured to deflect part of the first beam to        form a second beam that back-illuminates the sample in an        oblique angle;    -   (j) an absorber configured to absorb a remaining part of the        first beam that has a divergence angle.-   B2. The adaptor of embodiment B1, wherein the lens is positioned on    a front-side of the sample and the mirror is positioned to obliquely    illuminate the sample from a back-side of the sample, wherein the    oblique angle is larger than a collecting angle of the lens.-   B3. The adaptor of embodiment B1, wherein the mirror and the optical    absorber are mounted on a common structure and tilted with respect    to one another.-   B4. The adaptor of embodiment B1, further comprising a wavelength    filter positioned between the sample and the camera to pass    fluorescence emitted by the sample in response to the oblique    illumination.-   B5. The adaptor of embodiment B1, further comprising a rubber door    to cover the sample receptacle slot to prevent ambient light from    entering the optical assembly and entering the camera.-   B6. The adaptor of embodiment B1, wherein the light source and the    camera are positioned on the same side of the hand-held electronic    device and at fixed distance to one another.-   B7. The adaptor of embodiment B6, wherein the hand-held electronic    device is a smart phone.-   B8. The adaptor of embodiment B1, wherein the sample card is    supported by a sample holder comprising a planar structure, and    wherein the receptacle sample slot is configured to position the    planar structure to extend partially into a path of illumination    light from the light source to couple illumination light into the    planar structure.-   B9. The adaptor of embodiment B8, wherein the receptacle slot is    configured to position the planar structure such that the path of    illumination light is incident on an edge of the planar structure,    wherein the edge extends along a plane that is normal to a plane    comprising the field of view.-   B10. The adaptor of embodiment B8, wherein the mirror is arranged to    reflect the light to partially obliquely illuminate the sample from    a back side of the planar structure and to partially illuminate an    edge of the planar structure to couple illumination light into the    planar structure.-   B11 The adaptor of embodiment B8, wherein the planar structure is    configured to waveguide the coupled illumination light to the sample    to illuminate the sample and cause the sample to emit fluorescence.-   B12. A method for analyzing an analyte in a liquid sample,    comprising:

(a) obtaining the liquid sample;

(b) compressing at least part of the sample into a layer of uniformthickness with a sample card,

(c) inserting the sample card into an adaptor device, which isconfigured to be attached to an apparatus that comprises a light sourceand a camera;

(d) illuminating the sample with light from the light source, wherein

-   -   i. the light is filtered by an optical filter of the adapter        device to form a first beam with a specific wavelength range,        part of the first beam illuminating on the edge of the sample        card and travels in the sample card to illuminate the sample;    -   ii. part of the first beam is deflected by a mirror of the        adapter device to form a second beam that back-illuminates the        sample in an oblique angle; and    -   iii. a remaining part of the first beam that has a divergence        angle is absorbed by an absorber of the adapter device.

-   B13. The method of embodiment B2, further comprising:

(e) capturing images of the sample in the layer of uniform thicknesswith the camera;

(f) analyzing the images to enumerate the analyte in the images; and

(g) calculating the concentration of the analyte in the sample based onthe uniform thickness, a field of view of the camera, analyte number,and a predetermined correction factor;

wherein the field of view is the extent of the field in which the cameracaptures the images;

wherein the correction factor is determined by a miscount ratio, whichis dependent on the field of view, the uniform thickness, and propertiesof the analyte.

Additional Features:

-   C1. The device or method of any prior embodiments, wherein the    liquid sample is a blood sample.-   C2. The device or method of any prior embodiments, wherein the    analyte is white blood cells (WBC).-   C3. The device or method of any prior embodiments, wherein the    analyte is a WBC subtype.-   C4. The device or method of any prior embodiments, wherein the    analyte is neutrophils, eosinophils, basophils, lymphocytes, or    monocytes.-   C5. The device or method of any prior embodiments, wherein the    analyte is marked with fluorescence.-   C6. The device or method of any prior embodiments, wherein the    uniform thickness is in the range of 5 to 30 um.-   C7. The device or method of any prior embodiments, wherein the    uniform thickness is in the range of 8 to 12 um.-   C8. The device or method of any prior embodiments, wherein the    uniform thickness is around 10 um.-   C9. The device or method of any prior embodiments, wherein the field    of view (FOV) is equal to or larger than 4 mm².-   C10. The device or method of any prior embodiments, wherein the    field of view (FOV) is equal to or larger than 16 mm².-   C11. The device or method of any prior embodiments, wherein the    field of view (FOV) is equal to or larger than 36 mm².-   C12. The device or method of any prior embodiments, wherein the    field of view (FOV) is equal to or larger than 64 mm².-   C13. The device or method of any prior embodiments, wherein the    field of view (FOV) is equal to or larger than 100 mm².-   C14. The device or method of any prior embodiments, wherein the    correction factor is selected with the chart:

Sample Thickness Correction Factor 2 1 3 1 5 1.1 10 1.3 30 2.0

-   C15. The device or method of any prior embodiments, wherein the    analyte is marked with fluorescence and the wavelength range of the    first beam matches the excitation wavelength of the fluorescence    marking the analyte.-   C15. The device or method of any prior embodiments, wherein the    adaptor device further comprises a housing member.-   C16. The device or method of any prior embodiments, wherein the    adaptor device further comprises a lever, which can be inserted into    or extracted from the housing member.-   C17. The device or method of any prior embodiments, wherein the    mirror and the absorber are mounted on the lever.-   C18. The device or method of any prior embodiments, wherein adaptor    device comprises a card slot that has a secured opening that allows    the insertion of the sample card and prevents ambient light from    entering the card slot.

WBC Analysis Device

-   AA1. A device for analyzing white blood cells in a blood sample,    comprising:

a first plate, a second plate, and spacers, wherein:

-   -   i. the plates are movable relative to each other into different        configurations;    -   ii. one or both plates are flexible;    -   iii. each of the plates comprises an inner surface that has a        sample contact area for contacting a blood sample;    -   iv. one or both of the plates comprise the spacers that are        permanently fixed on the sample contact area of a respective        plate;    -   v. the spacers have:        -   (a) a predetermined substantially uniform height that has a            value selected in the range of 2 um to 30 um,        -   (b) a shape of pillar with substantially uniform            cross-section and a flat top surface;        -   (c) a ratio of the width to the height equal to or larger            than one;        -   (d) a predetermined, fixed, non-random, inter-spacer            distance that is in the range of 10 um to 200 um (micron);        -   (e) a filling factor of equal to 1% or larger, wherein the            filling factor is the ratio of the spacer contact area (on            the plate) to the total plate area; and        -   (f) the filling factor multiplies the Young's modulus of the            spacer is equal to 2 MPa or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart, the spacingbetween the plates is not regulated by the spacers, and the sample isdeposited on one or both of the plates;

wherein another of the configurations is a closed configuration which isconfigured after the sample is deposited in the open configuration; andin the closed configuration: at least part of the sample is compressedby the two plates into a layer of highly uniform thickness and issubstantially stagnant relative to the plates, wherein the uniformthickness of the layer is confined by the sample contact areas of thetwo plates and is regulated by the plates and the spacers.

-   AA2. A device for analyzing white blood cells in a blood sample,    comprising:

a first plate, a second plate, spacers, and adaptor wherein:

-   -   i. the plates are movable relative to each other into different        configurations;    -   ii. one or both plates are flexible;    -   iii. each of the plates comprises an inner surface that has a        sample contact area for contacting a fluidic sample;    -   iv. one or both of the plates comprise the spacers that are        permanently fixed on the sample contact area of a respective        plate;    -   v. the spacers have:        -   (a) a predetermined substantially uniform height that has a            value selected in the range of 2 um to 30 um,        -   (b) a shape of pillar with substantially uniform            cross-section and a flat top surface;        -   (c) a ratio of the width to the height equal or larger than            one;        -   (e) a predetermined fixed, non-random, inter-spacer distance            that is in the range of 10 um to 200 um;        -   (e) a filling factor of equal to 1% or larger, wherein the            filling factor is the ratio of the spacer contact area (on            the plate) to the total plate area; and        -   (f) the filling factor multiplies the Young's modulus of the            spacer is equal to 2 MPa or larger;    -   vi. the adaptor comprises: (a) a housing, (b) an attachment        member on the housing that allows the adaptor to be attached to        a mobile phone with a camera, (c) a slot in the housing that        allows (1) the plates in a closed configuration to slide into        the slot and (2) when the plates are in the slot, at least a        part of the sample area is less than 2 cm away from the outer        surface of the camera, and (d) an optical system in the housing        configured to have at least a part of the sample contact area be        imaged by the camera;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart, the spacingbetween the plates is not regulated by the spacers, and the sample isdeposited on one or both of the plates;

wherein another of the configurations is a closed configuration which isconfigured after the sample is deposited in the open configuration; andin the closed configuration: at least part of the sample is compressedby the two plates into a layer of highly uniform thickness and issubstantially stagnant relative to the plates, wherein the uniformthickness of the layer is confined by the sample contact areas of thetwo plates and is regulated by the plates and the spacers.

-   AA3. A method for analyzing white blood cells in a blood sample,    comprising:

(a) obtaining a blood sample;

(b) obtaining a device of AA1 or AA2;

(c) depositing the blood sample on one or both of the plates when theplates are configured in the open configuration,

(d) after (c), forcing the two plates into a closed configuration; and

(e) capturing images of sample in the layer of uniform thickness whilethe plates are the closed configuration; and

(f) analyzing the images to determine the concentration of white bloodcells in the sample.

-   AA4 A method for white blood cell and sub-type (including    neutrophils, eosinophils, basophils, lymphocytes, and monocytes)    counting using a single device, comprising:

(a) obtaining a blood sample;

(b) obtaining the device of any prior embodiments, wherein the spacerheight is 5 um to 15 um,

(c) depositing the blood sample on one or both of the plates when theplates are configured in an open configuration;

(d) after (c), forcing the two plates into a closed configuration;

(e) capturing images of the sample in the layer of uniform thicknesswhile the plates are the closed configuration; and

(f) analyzing the images to determine the respective number of whiteblood cells, neutrophils, lymphocytes, monocytes, eosinophils andbasophils, through the counting of the cell number in the image and theanalysis of the fluorescence color and shape for each white blood cell.

-   BB1. The device or method of any prior embodiments, wherein the    blood sample is undiluted.-   BB2. The device or method of any prior embodiments, wherein the    staining and shape of white blood cell provide fluorescence color    and dimension distinguish of white blood cell and its subtypes.-   BB3. The device or method of any prior embodiments, wherein the    device further comprises, on one or both plates, multi reagent    layers including anti-conglutination, cell lysing, cell staining,    release time control material layers, or their combinations.-   CC1. The device or method of any prior embodiments, wherein the    pillar height is in the range of 5 to 15 um,-   CC2. The device or method of any prior embodiments, wherein the    pillar height is in the range of 8 to 12 um,-   CC3. The device or method of any prior embodiments, wherein the    pillar height is around 10 um.-   CC4. The device or method of any prior embodiments, wherein the    device is configured to count the white blood cells.-   CC5. The device or method of any prior embodiments, wherein the    device is configured to count the white blood cells sub-types    (including neutrophils, eosinophils, basophils, lymphocytes, and    monocytes),-   CC6. The device or method of any prior embodiments, wherein spacer    height is in the range of 7.5 um to 10.5 um.-   CC7. The device or method of any prior embodiments, wherein spacer    height is in the range of 9.5 um to 12.5 um.-   CC8. The device or method of any prior embodiments, wherein spacer    height is in the range of 11.5 um to 13.5 um.-   CC9. The device or method of any prior embodiments, wherein spacer    height is in the range of 12.5 um to 14.5 um.-   CC10. The device or method of any prior embodiments, wherein spacer    height is in the range of 13.5 um to 16 um.-   CC11. The device or method of any prior embodiments, wherein a    preferred field of view for counting and differentiating WBCs is 0.1    mm², 10 mm², 50 mm², 100 mm² or a range between any two of the    values;-   CC12. The device or method of any prior embodiments, wherein when    the gap size of device is 10 um, the FoV is larger than 36 mm²,    thereby the WBC counting and differentiate accuracy is less than 5%.-   CC13. The device or method of any prior embodiments, wherein when    the gap size of device is 10 um, the FoV is larger than 16 mm²,    thereby the WBC counting and differentiate accuracy is less than    10%.-   CC14. The device or method of any prior embodiments, wherein when    the gap size of device is 10 um, the FoV is larger than 2 mm²,    thereby the WBC counting and differentiate accuracy is less than    20%.-   CC15. The device or method of any prior embodiments, wherein a field    of view is 0.1 mm² to 10 mm², preferred gap size of device is in the    range of 10 um to 30 um, 30 um to 50 um, thereby the counting and    differentiate accuracy is less than 10%.-   CC16. The device or method of any prior embodiments, wherein field    of view is 0.1 mm² to 10 mm², preferred gap size of device is in the    range of 10 um to 30 um, thereby the counting and differentiate    accuracy is less than 20%.-   CC17. The device or method of any prior embodiments, wherein field    of view is 10 mm² to 50 mm², preferred gap size of device is in the    range of 5 um to 10 um, 10 um to 30 um, thereby the counting and    differentiate accuracy is less than 10%.-   CC18. The device or method of any prior embodiments, wherein field    of view is 10 mm² to 50 mm², preferred gap size of device is in the    range of 2 um to 5 um, 5 um to 10 um, 10 um to 30 um, thereby the    counting and differentiate accuracy is less than 20%.-   CC19. The device or method of any prior embodiments, wherein field    of view is field of view of 50 mm² to 100 mm², preferred gap size of    device is in the range of 2 um to 5 um, 5 um to 10 um, 10 um to 30    um, 30 um to 50 um thereby the counting and differentiate accuracy    is less than 10%.-   CC20. The device or method of any prior embodiments, wherein the    spacer has a height in the range of 2 um to 5 um, thereby the WBCs    missing counting is less than 15%.-   CC21. The device or method of any prior embodiments, wherein the    spacer has a height in the range of 2 um to 5 um, 5 um to 10 um,    thereby the WBCs missing counting is less than 30%.-   CC22. The device or method of any prior embodiments, wherein the    spacer has a height of preferred range of 2 um to 5 um, 5 um to 10    um, 10 um to 30 um thereby the WBCs missing counting is less than    60%.-   CC23. The device or method of any prior embodiments, wherein the    sample to phone lens distance is in the range of 2 mm to 5 mm.-   CC24. The device or method of any prior embodiments, wherein the    sample to phone lens distance is in the range of 4 mm to 7 mm.-   CC25. The device or method of any prior embodiments, wherein the    sample to phone lens distance is in the range of 6 mm to 9 mm.-   CC26. The device or method of any prior embodiments, wherein the    sample to phone lens distance is in the range of 8 mm to 11 mm.-   CC27. The device or method of any prior embodiments, wherein the    sample to phone lens distance is in the range of 10 mm to 13 mm.-   CC28. The device or method of any prior embodiments, wherein the    sample to phone lens distance is in the range of 12 mm to 15 mm.

Imaging Analysis by Artificial Intelligence and Machine Learning

In certain embodiments of the present disclosure, the sample depositionis a deposition directly from a subject to the plate without using anytransferring devices. In certain embodiments, during the deposition, theamount of the sample deposited on the plate is unknown.

In certain embodiments, the method further comprises an analyzing thatanalyze the sample. In certain embodiments, the analyzing comprisescalculating the volume of a relevant sample volume by measuring thelateral area of the relevant sample volume and calculating the volumefrom the lateral area and the predetermined spacer height. In certainembodiments, the pH value at location of a sample that is between thetwo plates in a closed configuration is determined by the volume of thelocation and by analyzing an image(s) taken from that location. Incertain embodiments, the determination by analyzing an image usesartificial intelligence and machine learning

Artificial Intelligence and/or Machine Learning to Improve Imaging

In certain embodiments of the present invention, the images taken duringan assay operation and/or the samples measured by an assay are analyzedby artificial intelligence and machine learning. The samples include,but not limited to, medical samples, biology samples, environmentalsamples and chemistry samples.

In certain embodiments of the present invention, the sample is held by aQMAX device. The QMAX device together with imaging plus artificialintelligence and/or machine learning can overcome certain limitations inprior arts.

One important aspect of the present invention is to provide a machinelearning framework to enhance the functionality, application scope andthe accuracy in assaying using QMAX device, especially when a computerprogram is used.

In certain embodiments of the present invention, a device and a methodfor assaying sample and/or assay operation (e.g. tracking labelidentification) that utilizes QMAX together with imaging plus a machinelearning and/or artificial intelligence comprises:

-   -   (1) using a QMAX device that has an auxiliary structure in the        form of pillars to precisely control the distribution and volume        of the sample in assaying, wherein the sample for assaying is        loaded into the QMAX device and is kept between the two parallel        plates on the QMAX device with an upper plate being transparent        for imaging by an imager;    -   (2) the gap between the two parallel plates in the QMAX device        is spaced narrowly—with the distance of the gap being        proportional to the size of the analytes to be assayed—by which        the analytes in the sample form a single layer between the said        plates that can be imaged by an imager on the QMAX device;    -   (3) the sample volume corresponding to the AoI        (area-of-interest) on the upper plate of the QMAX device can be        precisely characterized by AoI and the gap—because of the        uniformity of the gap between the plates in the QMAX device;    -   (4) the image on the sample for assaying sandwiched between the        AoI x gap in the QMAX device is a pseudo-2D image, because it        has the appearance of a 2D image, but it is an image of a 3D        sample with its depth being known priori or characterized        through other means;    -   (5) the captured pseudo-2D sample image taken over the AoI of        the QMAX device can characterize the location of the analytes,        color, shape, counts, and concentration of the analytes in the        sample for assaying;    -   (6) based on abovementioned properties, the captured pseudo-2D        image of QMAX device for assaying is amendable to a machine        learning framework that applies to analyte detection,        localization, identification, segmentation, counting, etc. for        assaying in various applications; or    -   (7) any combination of thereof.

In certain embodiments of the present invention, a machine learningframework for QMAX based devices are implemented into a device that iscapable of running an algorithms such as deep learning todiscriminatively locate, identify, segment and count analytes (e.g.blood cells) based on the pseudo-2D image captured by the QMAX imager.

In certain embodiments of the present invention, the machine learningimproves the images captured by the imager on the QMAX device andreduces the effects of noise and artifacts—including and not limited toair bobbles, dusts, shadows, and pillars.

In certain embodiments of the present invention, the training of machinelearning uses the spacers of the QMAX card to reduce the data size oftraining set.

Approach 1—Deep Learning Approach

In certain embodiments, deep learning is used, whereinthe analytedetection and localization workflow consists of two stages, training andprediction.

(i) Training Stage

At the training stage of the present invention, training data withannotation is fed into a convolutional neural network. Convolutionalneural network is a specialized neural network for processing data thathas a grid-like, feed forward and layered network topology. Examples ofthe data include time-series data, which can be thought of as a 1D gridtaking samples at regular time intervals, and image data, which can bethought of as a 2D grid of pixels. Convolutional networks have beensuccessful in practical applications. The name “convolutional neuralnetwork” indicates that the network employs a mathematical operationcalled convolution. Convolution is a specialized kind of linearoperation. Convolutional networks are simply neural networks that useconvolution in place of general matrix multiplication in at least one oftheir layers.

In training the machine learning model in some embodiments of thepresent invention, it receives one or multiple images of samples thatcontain the analytes taken by the imager over the sample holding QMAXdevice as training data. Training data are annotated for analytes to beassayed, wherein the annotations indicate whether or not analytes are inthe training data and where they locate in the image. Annotation can bedone in the form of tight bounding boxes which fully contains theanalyte, or center locations of analytes. In the latter case, centerlocations are further converted into circles covering analytes or aGaussian kernel in a point map.

When the size of training data is large, training machine learning modelpresents two challenges: annotation (usually done by human) is timeconsuming, and the training is computationally expensive. To overcomethese challenges, one can partition the training data into patches ofsmall size, then annotate and train on these patches, or a portion ofthese patches. The term “machine learning” refers to algorithms, systemsand apparatus in the field of artificial intelligence that often usestatistical techniques and artificial neural network trained from datawithout being explicitly programmed.

In some embodiments of the present invention, the annotated images arefed to the machine learning (ML) training module, and the model trainerin the machine learning module will train a ML model from the trainingdata (annotated sample images). The input data will be fed to the modeltrainer in multiple iterations until certain stopping criterion issatisfied. The output of the ML training module is a ML model—acomputational model that is built from a training process in the machinelearning from the data that gives computer the capability to performcertain tasks (e.g. detect and classify the objects) on its own.

The trained machine learning model is applied during the predication (orinference) stage by the computer. Examples of machine learning modelsinclude ResNet, DenseNet, etc. which are also named as “deep learningmodels” because of the depth of the connected layers in their networkstructure. In some embodiments, the Caffe library with fullyconvolutional network (FCN) was used for model training and predication,and other convolutional neural network architecture and library can alsobe used, such as TensorFlow.

The training stage generates a model that will be used in the predictionstage. The model can be repeatedly used in the prediction stage forassaying the input. Thus, the computing unit only needs access to thegenerated model. It does not need access to the training data, norrequiring the training stage to be run again on the computing unit.

(ii) Prediction Stage

In the predication/inference stage, a detection component is applied tothe input image, and an input image is fed into the predication(inference) module preloaded with a trained model generated from thetraining stage. The output of the prediction stage can be bounding boxesthat contain the detected analytes with their center locations or apoint map indicating the location of each analyte, or a heatmap thatcontains the information of the detected analytes.

When the output of the prediction stage is a list of bounding boxes, thenumber of analytes in the image of the sample for assaying ischaracterized by the number of detected bounding boxes. When the outputof the prediction stage is a point map, the number of analytes in theimage of the sample for assaying is characterized by the integration ofthe point map. When the output of the prediction is a heatmap, alocalization component is used to identify the location and the numberof detected analytes is characterized by the entries of the heatmap.

One embodiment of the localization algorithm is to sort the heatmapvalues into a one-dimensional ordered list, from the highest value tothe lowest value. Then pick the pixel with the highest value, remove thepixel from the list, along with its neighbors. Iterate the process topick the pixel with the highest value in the list, until all pixels areremoved from the list.

In the detection component using heatmap, an input image, along with themodel generated from the training stage, is fed into a convolutionalneural network, and the output of the detection stage is a pixel-levelprediction, in the form of a heatmap. The heatmap can have the same sizeas the input image, or it can be a scaled down version of the inputimage, and it is the input to the localization component. We disclose analgorithm to localize the analyte center. The main idea is toiteratively detect local peaks from the heatmap. After the peak islocalized, we calculate the local area surrounding the peak but withsmaller value. We remove this region from the heatmap and find the nextpeak from the remaining pixels. The process is repeated only all pixelsare removed from the heatmap.

In certain embodiments, the present invention provides the localizationalgorithm to sort the heatmap values into a one-dimensional orderedlist, from the highest value to the lowest value. Then pick the pixelwith the highest value, remove the pixel from the list, along with itsneighbors. Iterate the process to pick the pixel with the highest valuein the list, until all pixels are removed from the list.

Algorithm GlobalSearch (heatmap) Input: heatmap Output: loci loci ←{ }sort(heatmap) while (heatmap is not empty) { s ← pop(heatmap) D ← {diskcenter as s with radius R} heatmap = heatmap \ D // remove D from theheatmap add s to loc}

After sorting, heatmap is a one-dimensional ordered list, where theheatmap value is ordered from the highest to the lowest. Each heatmapvalue is associated with its corresponding pixel coordinates. The firstitem in the heatmap is the one with the highest value, which is theoutput of the pop(heatmap) function. One disk is created, where thecenter is the pixel coordinate of the one with highest heatmap value.Then all heatmap values whose pixel coordinates resides inside the diskis removed from the heatmap. The algorithm repeatedly pops up thehighest value in the current heatmap, removes the disk around it, tillthe items are removed from the heatmap.

In the ordered list heatmap, each item has the knowledge of theproceeding item, and the following item. When removing an item from theordered list, we make the following changes:

-   -   Assume the removing item is x_(r), its proceeding item is x_(p),        and its following item is x_(f).    -   For the proceeding item x_(p), re-define its following item to        the following item of the removing item. Thus, the following        item of x_(p) is now x_(f).    -   For the removing item x_(r), un-define its proceeding item and        following item, which removes it from the ordered list.    -   For the following item x_(f), re-define its proceeding item to        the proceeding item of the removed item. Thus, the proceeding        item of x_(f) is now x_(p).

After all items are removed from the ordered list, the localizationalgorithm is complete. The number of elements in the set loci will bethe count of analytes, and location information is the pixel coordinatefor each s in the set loci.

Another embodiment searches local peak, which is not necessary the onewith the highest heatmap value. To detect each local peak, we start froma random starting point, and search for the local maximal value. Afterwe find the peak, we calculate the local area surrounding the peak butwith smaller value. We remove this region from the heatmap and find thenext peak from the remaining pixels. The process is repeated only allpixels are removed from the heatmap.

Algorithm LocalSearch (s, heatmap) Input: s: starting location (x, y)heatmap Output: s: location of local peak. We only consider pixels ofvalue > 0. Algorithm Cover (s, heatmap) Input: s: location of localpeak. heatmap: Output: cover: a set of pixels covered by peak:

This is a breadth-first-search algorithm starting from s, with onealtered condition of visiting points: a neighbor p of the currentlocation q is only added to cover if heatmap[p]>0 andheatmap[p]<=heatmap[q]. Therefore, each pixel in cover has anon-descending path leading to the local peak s.

Algorithm Localization (heatmap) Input: heatmap Output: loci loci ←{ }pixels ←{all pixels from heatmap} while pixels is not empty { s ←anypixel from pixels s ←LocalSearch(s, heatmap) // s is now local peakprobe local region of radius R surrounding s for better local peak r←Cover(s, heatmap) pixels ← pixels \ r // remove all pixels in cover adds to loci

In certain embodiments, the image analysis comprising a Combination ofDeep Learning and Computer Vision Approach, wherein I the detection andlocalization are realized by computer vision algorithms, and aclassification is realized by deep learning algorithms, wherein thecomputer vision algorithms detect and locate possible candidates ofanalytes, and the deep learning algorithm classifies each possiblecandidate as a true analyte and false analyte. The location of all trueanalyte (along with the total count of true analytes) will be recordedas the output.

Detection. The computer vision algorithm detects possible candidatebased on the characteristics of analytes, including but not limited tointensity, color, size, shape, distribution, etc. A pre-processingscheme can improve the detection. Pre-processing schemes includecontrast enhancement, histogram adjustment, color enhancement,de-nosing, smoothing, de-focus, etc. After pre-processing, the inputimage is sent to a detector. The detector tells the existing of possiblecandidate of analyte and gives an estimate of its location. Thedetection can be based on the analyte structure (such as edge detection,line detection, circle detection, etc.), the connectivity (such as blobdetection, connect components, contour detection, etc.), intensity,color, shape using schemes such as adaptive thresholding, etc.

Localization After detection, the computer vision algorithm locates eachpossible candidate of analytes by providing its boundary or a tightbounding box containing it. This can be achieved through objectsegmentation algorithms, such as adaptive thresholding, backgroundsubtraction, floodfill, mean shift, watershed, etc. Very often, thelocalization can be combined with detection to produce the detectionresults along with the location of each possible candidates of analytes.

Classification, the deep learning algorithms, such as convolutionalneural networks, achieve start-of-the-art visual classification. Weemploy deep learning algorithms for classification on each possiblecandidate of analytes. Various convolutional neural network can beutilized for analyte classification, such as VGGNet, ResNet, MobileNet,DenseNet, etc.

Given each possible candidate of analyte, the deep learning algorithmcomputes through layers of neurons via convolution filters andnon-linear filters to extract high-level features that differentiateanalyte against non-analytes. A layer of fully convolutional networkwill combine high-level features into classification results, whichtells whether it is a true analyte or not, or the probability of beingan analyte.

Flat Top of Pillar Spacers

In certain embodiments of the present invention, the spacers are pillarsthat have a flat top and a foot fixed on one plate, wherein the flat tophas a smoothness with a small surface variation, and the variation isless than 5, 10 nm, 20 nm, 30 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm,500 nm, 600 nm, 700 nm, 800 nm, 1000 nm, or in a range between any twoof the values. A preferred flat pillar top smoothness is that surfacevariation of 50 nm or less.

Furthermore, the surface variation is relative to the spacer height andthe ratio of the pillar flat top surface variation to the spacer heightis less than 0.5%, 1%, 3%, 5%, 7%, 10%, 15%, 20%, 30%, 40%, or in arange between any two of the values. A preferred flat pillar topsmoothness has a ratio of the pillar flat top surface variation to thespacer height is less than 2%, 5%, or 10%.

Sidewall Angle of Pillar Spacers

In certain embodiments of the present invention, the spacers are pillarsthat have a sidewall angle. In some embodiments, the sidewall angle isless than 5 degree (measured from the normal of a surface), 10 degree,20 degree, 30 degree, 40 degree, 50 degree, 70 degree, or in a rangebetween any two of the values. In a preferred embodiment, the sidewallangle is less than 5 degree, 10 degree, or 20 degree.

Formation of Uniform Thin Fluidic Layer by an Imprecise Force Pressing

In certain embodiment of the present invention, a uniform thin fluidicsample layer is formed by using a pressing with an imprecise force. Theterm “imprecise pressing force” without adding the details and thenadding a definition for imprecise pressing force. As used herein, theterm “imprecise” in the context of a force (e.g. “imprecise pressingforce”) refers to a force that

(a) has a magnitude that is not precisely known or precisely predictableat the time the force is applied; (b) has a pressure in the range of0.01 kg/cm² (centimeter square) to 100 kg/cm², (c) varies in magnitudefrom one application of the force to the next; and (d) the imprecision(i.e. the variation) of the force in (a) and (c) is at least 20% of thetotal force that actually is applied.

An imprecise force can be applied by human hand, for example, e.g., bypinching an object together between a thumb and index finger, or bypinching and rubbing an object together between a thumb and indexfinger.

In some embodiments, the imprecise force by the hand pressing has apressure of 0.01 kg/cm2, 0.1 kg/cm2, 0.5 kg/cm2, 1 kg/cm2, 2 kg/cm2,kg/cm2, 5 kg/cm2, 10 kg/cm2, 20 kg/cm2, 30 kg/cm2, 40 kg/cm2, 50 kg/cm2,60 kg/cm2, 100 kg/cm2, 150 kg/cm2, 200 kg/cm2, or a range between anytwo of the values; and a preferred range of 0.1 kg/cm2 to 0.5 kg/cm2,0.5 kg/cm2 to 1 kg/cm2, 1 kg/cm2 to 5 kg/cm2, 5 kg/cm2 to 10 kg/cm2(Pressure).

Spacer Filling Factor.

The term “spacer filling factor” or “filling factor” refers to the ratioof the spacer contact area to the total plate area”, wherein the spacercontact area refers, at a closed configuration, the contact area thatthe spacer's top surface contacts to the inner surface of a plate, andthe total plate area refers the total area of the inner surface of theplate that the flat top of the spacers contact. Since there are twoplates and each spacer has two contact surfaces each contacting oneplate, the filling fact is the filling factor of the smallest.

For example, if the spacers are pillars with a flat top of a squareshape (10 um×10 um), a nearly uniform cross-section and 2 um tall, andthe spacers are periodic with a period of 100 um, then the filing factorof the spacer is 1%. If in the above example, the foot of the pillarspacer is a square shape of 15 um×15 um, then the filling factor isstill 1% by the definition.

Example of Present Embodiments IDŜ4/hE

In certain embodiments of the present disclosure, a device for forming athin fluidic sample layer with a uniform predetermined thickness bypressing can comprise a first plate. In certain embodiments of thepresent disclosure, a device for forming a thin fluidic sample layerwith a uniform predetermined thickness by pressing can comprise a secondplate. In certain embodiments of the present disclosure, a device forforming a thin fluidic sample layer with a uniform predeterminedthickness by pressing can comprise spacers. In certain embodiments, theplates are movable relative to each other into different configurations.In certain embodiments, one or both plates are flexible. In certainembodiments, each of the plates comprises an inner surface that has asample contact area for contacting a fluidic sample. In certainembodiments, each of the plates comprises, on its respective outersurface, a force area for applying a pressing force that forces theplates together. In certain embodiments, one or both of the platescomprise the spacers that are permanently fixed on the inner surface ofa respective plate. In certain embodiments, the spacers have apredetermined substantially uniform height that is equal to or less than200 microns, and a predetermined fixed inter-spacer-distance. In certainembodiments, the fourth power of the inter-spacer-distance (ISD) dividedby the thickness (h) and the Young's modulus (E) of the flexible plate(ISD⁴/(hE)) is 5×10⁶ um³/GPa or less. In certain embodiments, at leastone of the spacers is inside the sample contact area. In certainembodiments, one of the configurations is an open configuration, inwhich: the two plates are partially or completely separated apart, thespacing between the plates is not regulated by the spacers, and thesample is deposited on one or both of the plates. In certainembodiments, another of the configurations is a closed configurationwhich is configured after the sample is deposited in the openconfiguration and the plates are forced to the closed configuration byapplying the pressing force on the force area; and in the closedconfiguration: at least part of the sample is compressed by the twoplates into a layer of highly uniform thickness and is substantiallystagnant relative to the plates, wherein the uniform thickness of thelayer is confined by the sample contact areas of the two plates and isregulated by the plates and the spacers.

In certain embodiments of the present disclosure, a method of forming athin fluidic sample layer with a uniform predetermined thickness bypressing can comprise obtaining a device of the present disclosure. Incertain embodiments of the present disclosure, a method of forming athin fluidic sample layer with a uniform predetermined thickness bypressing can comprise depositing a fluidic sample on one or both of theplates when the plates are configured in an open configuration. Incertain embodiments, the open configuration is a configuration in whichthe two plates are partially or completely separated apart and thespacing between the plates is not regulated by the spacers. In certainembodiments of the present disclosure, a method of forming a thinfluidic sample layer with a uniform predetermined thickness by pressingcan comprise forcing the two plates into a closed configuration, inwhich: at least part of the sample is compressed by the two plates intoa layer of substantially uniform thickness, wherein the uniformthickness of the layer is confined by the sample contact surfaces of theplates and is regulated by the plates and the spacers.

In certain embodiments of the present disclosure, a device for analyzinga fluidic sample can comprise a first plate. In certain embodiments ofthe present disclosure, a device for analyzing a fluidic sample cancomprise a second plate. In certain embodiments of the presentdisclosure, a device for analyzing a fluidic sample can comprisespacers. In certain embodiments, the plates are movable relative to eachother into different configurations. In certain embodiments, one or bothplates are flexible. In certain embodiments, each of the plates has, onits respective inner surface, a sample contact area for contacting afluidic sample. In certain embodiments, one or both of the platescomprise the spacers and the spacers are fixed on the inner surface of arespective plate. In certain embodiments, the spacers have apredetermined substantially uniform height that is equal to or less than200 microns, and the inter-spacer-distance is predetermined. In certainembodiments, the Young's modulus of the spacers multiplied by thefilling factor of the spacers is at least 2 MPa. In certain embodiments,at least one of the spacers is inside the sample contact area. Incertain embodiments, one of the configurations is an open configuration,in which: the two plates are partially or completely separated apart,the spacing between the plates is not regulated by the spacers, and thesample is deposited on one or both of the plates. In certainembodiments, another of the configurations is a closed configurationwhich is configured after the sample is deposited in the openconfiguration; and in the closed configuration: at least part of thesample is compressed by the two plates into a layer of highly uniformthickness, wherein the uniform thickness of the layer is confined by thesample contact surfaces of the plates and is regulated by the plates andthe spacers.

In certain embodiments of the present disclosure, a method of forming athin fluidic sample layer with a uniform predetermined thickness bypressing can comprise obtaining a device of the present disclosure. Incertain embodiments of the present disclosure, a method of forming athin fluidic sample layer with a uniform predetermined thickness bypressing can comprise depositing a fluidic sample on one or both of theplates when the plates are configured in an open configuration. Incertain embodiments, the open configuration is a configuration in whichthe two plates are partially or completely separated apart and thespacing between the plates is not regulated by the spacers. In certainembodiments of the present disclosure, a method of forming a thinfluidic sample layer with a uniform predetermined thickness by pressingcan comprise forcing the two plates into a closed configuration. Incertain embodiments, at least part of the sample is compressed by thetwo plates into a layer of substantially uniform thickness, wherein theuniform thickness of the layer is confined by the sample contactsurfaces of the plates and is regulated by the plates and the spacers.

In certain embodiments of the present disclosure, a device for analyzinga fluidic sample can comprise a first plate. In certain embodiments ofthe present disclosure, a device for analyzing a fluidic sample cancomprise a second plate. In certain embodiments, the plates are movablerelative to each other into different configurations. In certainembodiments, one or both plates are flexible. In certain embodiments,each of the plates has, on its respective surface, a sample contact areafor contacting a sample that contains an analyte. In certainembodiments, one or both of the plates comprise spacers that arepermanently fixed to a plate within a sample contact area, wherein thespacers have a predetermined substantially uniform height and apredetermined fixed inter-spacer distance that is at least about 2 timeslarger than the size of the analyte, up to 200 um, and wherein at leastone of the spacers is inside the sample contact area. In certainembodiments, one of the configurations is an open configuration, inwhich: the two plates are separated apart, the spacing between theplates is not regulated by the spacers, and the sample is deposited onone or both of the plates. In certain embodiments, another of theconfigurations is a closed configuration which is configured after thesample deposition in the open configuration; and in the closedconfiguration: at least part of the sample is compressed by the twoplates into a layer of highly uniform thickness, wherein the uniformthickness of the layer is confined by the sample contact surfaces of theplates and is regulated by the plates and the spacers.

In certain embodiments of the present disclosure, a method of forming athin fluidic sample layer with a uniform predetermined thickness bypressing can comprise obtaining a device of the present disclosure. Incertain embodiments of the present disclosure a method of forming a thinfluidic sample layer with a uniform predetermined thickness by pressingcan comprise depositing a fluidic sample on one or both of the plates;when the plates are configured in an open configuration, wherein theopen configuration is a configuration in which the two plates arepartially or completely separated apart and the spacing between theplates is not regulated by the spacers. In certain embodiments of thepresent disclosure a method of forming a thin fluidic sample layer witha uniform predetermined thickness by pressing can comprise forcing thetwo plates into a closed configuration, in which: at least part of thesample is compressed by the two plates into a layer of substantiallyuniform thickness, wherein the uniform thickness of the layer isconfined by the sample contact surfaces of the plates and is regulatedby the plates and the spacers.

In certain embodiments of the present disclosure, a device for forming athin fluidic sample layer with a uniform predetermined thickness bypressing can comprise a first plate. In certain embodiments of thepresent disclosure, a device for forming a thin fluidic sample layerwith a uniform predetermined thickness by pressing can comprise a secondplate. In certain embodiments of the present disclosure, a device forforming a thin fluidic sample layer with a uniform predeterminedthickness by pressing can comprise spacers. In certain embodiments, theplates are movable relative to each other into different configurations.In certain embodiments, one or both plates are flexible. In certainembodiments, each of the plates comprises, on its respective innersurface, a sample contact area for contacting and/or compressing afluidic sample. In certain embodiments, each of the plates comprises, onits respective outer surface, an area for applying a force that forcesthe plates together. In certain embodiments, one or both of the platescomprise the spacers that are permanently fixed on the inner surface ofa respective plate. In certain embodiments, the spacers have apredetermined substantially uniform height that is equal to or less than200 microns, a predetermined width, and a predetermined fixedinter-spacer-distance. In certain embodiments, a ratio of theinter-spacer-distance to the spacer width is 1.5 or larger. In certainembodiments, at least one of the spacers is inside the sample contactarea. In certain embodiments, one of the configurations is an openconfiguration, in which: the two plates are partially or completelyseparated apart, the spacing between the plates is not regulated by thespacers, and the sample is deposited on one or both of the plates. Incertain embodiments, another of the configurations is a closedconfiguration which is configured after the sample deposition in theopen configuration; and in the closed configuration: at least part ofthe sample is compressed by the two plates into a layer of highlyuniform thickness and is substantially stagnant relative to the plates,wherein the uniform thickness of the layer is confined by the samplecontact areas of the two plates and is regulated by the plates and thespacers.

In certain embodiments of the present disclosure, a method of forming athin fluidic sample layer with a uniform predetermined thickness bypressing with an imprecise pressing force can comprise obtaining adevice of the present disclosure. In certain embodiments of the presentdisclosure, a method of forming a thin fluidic sample layer with auniform predetermined thickness by pressing with an imprecise pressingforce can comprise obtaining a fluidic sample. In certain embodiments ofthe present disclosure, a method of forming a thin fluidic sample layerwith a uniform predetermined thickness by pressing with an imprecisepressing force can comprise depositing the sample on one or both of theplates; when the plates are configured in an open configuration, whereinthe open configuration is a configuration in which the two plates arepartially or completely separated apart and the spacing between theplates is not regulated by the spacers. In certain embodiments of thepresent disclosure, a method of forming a thin fluidic sample layer witha uniform predetermined thickness by pressing with an imprecise pressingforce can comprise forcing the two plates into a closed configuration,in which: at least part of the sample is compressed by the two platesinto a layer of substantially uniform thickness, wherein the uniformthickness of the layer is confined by the sample contact surfaces of theplates and is regulated by the plates and the spacers.

In certain embodiments, the spacers have a shape of pillar with a footfixed on one of the plates and a flat top surface for contacting theother plate. In certain embodiments, the spacers have a shape of pillarwith a foot fixed on one of the plates, a flat top surface forcontacting the other plate, substantially uniform cross-section. Incertain embodiments, the spacers have a shape of pillar with a footfixed on one of the plates and a flat top surface for contacting theother plate, wherein the flat top surface of the pillars has a variationin less than 10 nm. In certain embodiments, the spacers have a shape ofpillar with a foot fixed on one of the plates and a flat top surface forcontacting the other plate, wherein the flat top surface of the pillarshas a variation in less than 50 nm. In certain embodiments, the spacershave a shape of pillar with a foot fixed on one of the plates and a flattop surface for contacting the other plate, wherein the flat top surfaceof the pillars has a variation in less than 50 nm. In certainembodiments, the spacers have a shape of pillar with a foot fixed on oneof the plates and a flat top surface for contacting the other plate,wherein the flat top surface of the pillars has a variation in less than10 nm, 20 nm, 30 nm, 100 nm, 200 nm, or in a range of any two of thevalues.

In certain embodiments, the Young's modulus of the spacers multiplied bythe filling factor of the spacers is at least 2 MPa. In certainembodiments, the sample comprises an analyte and the predeterminedconstant inter-spacer distance is at least about 2 times larger than thesize of the analyte, up to 200 um. In certain embodiments, the samplecomprise an analyte, the predetermined constant inter-spacer distance isat least about 2 times larger than the size of the analyte, up to 200um, and the Young's modulus of the spacers multiplied by the fillingfactor of the spacers is at least 2 MPa.

In certain embodiments, a fourth power of the inter-spacer-distance(IDS) divided by the thickness (h) and the Young's modulus (E) of theflexible plate (ISD̂4/(hE)) is 5×10̂6 um̂3/GPa or less. In certainembodiments, a fourth power of the inter-spacer-distance (IDS) dividedby the thickness and the Young's modulus of the flexible plate(ISD̂4/(hE)) is 1×10̂6 um̂3/GPa or less. In certain embodiments, a fourthpower of the inter-spacer-distance (IDS) divided by the thickness andthe Young's modulus of the flexible plate (ISD̂4/(hE)) is 5×10̂5 um̂3/GPaor less. In certain embodiments, the Young's modulus of the spacersmultiplied by the filling factor of the spacers is at least 2 MPa, and afourth power of the inter-spacer-distance (IDS) divided by the thicknessand the Young's modulus of the flexible plate (ISD̂4/(hE)) is 1×10̂5um̂3/GPa or less. In certain embodiments, the Young's modulus of thespacers multiplied by the filling factor of the spacers is at least 2MPa, and a fourth power of the inter-spacer-distance (IDS) divided bythe thickness and the Young's modulus of the flexible plate (ISD̂4/(hE))is 1×10̂4 um̂3/GPa or less. In certain embodiments, the Young's modulus ofthe spacers multiplied by the filling factor of the spacers is at least20 MPa.

In certain embodiments of the present disclosure, the ratio of theinter-spacing distance of the spacers to the average width of the spaceris 2 or larger. In certain embodiments, the ratio of the inter-spacingdistance of the spacers to the average width of the spacer is 2 orlarger, and the Young's modulus of the spacers multiplied by the fillingfactor of the spacers is at least 2 MPa. In certain embodiments, theinter-spacer distance that is at least about 2 times larger than thesize of the analyte, up to 200 um. In certain embodiments, a ratio ofthe inter-spacer-distance to the spacer width is 1.5 or larger. Incertain embodiments, a ratio of the width to the height of the spacer is1 or larger. In certain embodiments, a ratio of the width to the heightof the spacer is 1.5 or larger. In certain embodiments, a ratio of thewidth to the height of the spacer is 2 or larger. In certainembodiments, a ratio of the width to the height of the spacer is largerthan 2, 3, 5, 10, 20, 30, 50, or in a range of any two the value.

In certain embodiments, a force that presses the two plates into theclosed configuration is an imprecise pressing force. In certainembodiments, a force that presses the two plates into the closedconfiguration is an imprecise pressing force provided by human hand. Incertain embodiments, the forcing of the two plates to compress at leastpart of the sample into a layer of substantially uniform thicknesscomprises a use of a conformable pressing, either in parallel orsequentially, an area of at least one of the plates to press the platestogether to a closed configuration, wherein the conformable pressinggenerates a substantially uniform pressure on the plates over the atleast part of the sample, and the pressing spreads the at least part ofthe sample laterally between the sample contact surfaces of the plates,and wherein the closed configuration is a configuration in which thespacing between the plates in the layer of uniform thickness region isregulated by the spacers; and wherein the reduced thickness of thesample reduces the time for mixing the reagents on the storage site withthe sample. In certain embodiments, the pressing force is an impreciseforce that has a magnitude which is, at the time that the force isapplied, either (a) unknown and unpredictable, or (b) cannot be knownand cannot be predicted within an accuracy equal or better than 20% ofthe average pressing force applied. In certain embodiments, the pressingforce is an imprecise force that has a magnitude which is, at the timethat the force is applied, either (a) unknown and unpredictable, or (b)cannot be known and cannot be predicted within an accuracy equal orbetter than 30% of the average pressing force applied. In certainembodiments, the pressing force is an imprecise force that has amagnitude which is, at the time that the force is applied, either (a)unknown and unpredictable, or (b) cannot be known and cannot bepredicted within an accuracy equal or better than 30% of the averagepressing force applied; and wherein the layer of highly uniformthickness has a variation in thickness uniform of 20% or less. Incertain embodiments, the pressing force is an imprecise force that has amagnitude which cannot, at the time that the force is applied, bedetermined within an accuracy equal or better than 30%, 40%, 50%, 70%,100%, 200%, 300%, 500%, 1000%, 2000%, or in a range between any of thetwo values.

In certain embodiments of the present disclosure, the flexible plate hasa thickness of in the range of 10 um to 200 um. In certain embodiments,the flexible plate has a thickness of in the range of 20 um to 100 um.In certain embodiments, the flexible plate has a thickness of in therange of 25 um to 180 um. In certain embodiments, the flexible plate hasa thickness of in the range of 200 um to 260 um. In certain embodiments,the flexible plate has a thickness of equal to or less than 250 um, 225um, 200 um, 175 um, 150 um, 125 um, 100 um, 75 um, 50 um, 25 um, 10 um,5 um, 1 um, or in a range between the two of the values. In certainembodiments, the sample has a viscosity in the range of 0.1 to 4 (mPas). In certain embodiments, the flexible plate has a thickness of in therange of 200 um to 260 um. In certain embodiments, the flexible platehas a thickness in the range of 20 um to 200 um and Young's modulus inthe range 0.1 to 5 GPa.

In certain embodiments of the present disclosure, the sample depositionis a deposition directly from a subject to the plate without using anytransferring devices. In certain embodiments, during the deposition, theamount of the sample deposited on the plate is unknown. In certainembodiments, the method further comprises an analyzing that analyze thesample. In certain embodiments, the analyzing comprises calculating thevolume of a relevant sample volume by measuring the lateral area of therelevant sample volume and calculating the volume from the lateral areaand the predetermined spacer height. In certain embodiments, the pHvalue at location of a sample that is between the two plates in a closedconfiguration is determined by the volume of the location and byanalyzing an image(s) taken from that location. In certain embodiments,the determination by analyzing an image uses artificial intelligence andmachine learning.

In certain embodiments, the analyzing step (e) comprises measuring: i.imaging, ii. luminescence selected from photoluminescence,electroluminescence, and electrochemiluminescence, iii. surface Ramanscattering, iv. electrical impedance selected from resistance,capacitance, and inductance, or v. any combination of i-iv. In certainembodiments, the analyzing comprises reading, image analysis, orcounting of the analyte, or a combination of thereof. In certainembodiments, the sample contains one or plurality of analytes, and oneor both plate sample contact surfaces comprise one or a plurality ofbinding sites that each binds and immobilize a respective analyte. Incertain embodiments, one or both plate sample contact surfaces compriseone or a plurality of storage sites that each stores a reagent orreagents, wherein the reagent(s) dissolve and diffuse in the sample. Incertain embodiments, one or both plate sample contact surfaces comprisesone or a plurality of amplification sites that are each capable ofamplifying a signal from the analyte or a label of the analyte when theanalyte or label is within 500 nm from an amplification site. In certainembodiments, i. one or both plate sample contact surfaces comprise oneor a plurality of binding sites that each binds and immobilize arespective analyte; or ii. one or both plate sample contact surfacescomprise, one or a plurality of storage sites that each stores a reagentor reagents; wherein the reagent(s) dissolve and diffuse in the sample,and wherein the sample contains one or plurality of analytes; or iii.one or a plurality of amplification sites that are each capable ofamplifying a signal from the analyte or a label of the analyte when theanalyte or label is 500 nm from the amplification site; or iv. anycombination of i to iii.

In certain embodiments, the liquid sample is a biological sampleselected from amniotic fluid, aqueous humour, vitreous humour, blood(e.g., whole blood, fractionated blood, plasma or serum), breast milk,cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph,perilymph, feces, breath, gastric acid, gastric juice, lymph, mucus(including nasal drainage and phlegm), pericardial fluid, peritonealfluid, pleural fluid, pus, rheum, saliva, exhaled breath condensates,sebum, semen, sputum, sweat, synovial fluid, tears, vomit, and urine.

In certain embodiments, the layer of uniform thickness in the closedconfiguration is less than 150 um. In certain embodiments, the pressingis provided by a pressured liquid, a pressed gas, or a conformalmaterial. In certain embodiments, the analyzing comprises counting cellsin the layer of uniform thickness. In certain embodiments, the analyzingcomprises performing an assay in the layer of uniform thickness. Incertain embodiments, In certain embodiments, the assay is a bindingassay or biochemical assay. In certain embodiments, the sample depositedhas a total volume less 0.5 uL. In certain embodiments, multiple dropsof sample are deposited onto one or both of the plates.

In certain embodiments, the inter-spacer distance is in the range of 1μm to 120 μm. In certain embodiments, the inter-spacer distance is inthe range of 120 μm to 50 μm. In certain embodiments, the inter-spacerdistance is in the range of 120 μm to 200 μm. In certain embodiments,the flexible plates have a thickness in the range of 20 um to 250 um andYoung's modulus in the range 0.1 to 5 GPa. In certain embodiments, for aflexible plate, the thickness of the flexible plate times the Young'smodulus of the flexible plate is in the range 60 to 750 GPa-um.

In certain embodiments, the layer of uniform thickness sample is uniformover a lateral area that is at least 1 mm². In certain embodiments, thelayer of uniform thickness sample is uniform over a lateral area that isat least 3 mm². In certain embodiments, the layer of uniform thicknesssample is uniform over a lateral area that is at least 5 mm². In certainembodiments, In certain embodiments, the layer of uniform thicknesssample is uniform over a lateral area that is at least 10 mm². Incertain embodiments, the layer of uniform thickness sample is uniformover a lateral area that is at least 20 mm². In certain embodiments, thelayer of uniform thickness sample is uniform over a lateral area that isin a range of 20 mm² to 100 mm². In certain embodiments, the layer ofuniform thickness sample has a thickness uniformity of up to +/−5% orbetter. In certain embodiments, the layer of uniform thickness samplehas a thickness uniformity of up to +/−10% or better. In certainembodiments, the layer of uniform thickness sample has a thicknessuniformity of up to +/−20% or better. In certain embodiments, the layerof uniform thickness sample has a thickness uniformity of up to +/−30%or better. In certain embodiments, the layer of uniform thickness samplehas a thickness uniformity of up to +/−40% or better. In certainembodiments, the layer of uniform thickness sample has a thicknessuniformity of up to +/−50% or better.

In certain embodiments, the spacers are pillars with a cross-sectionalshape selected from round, polygonal, circular, square, rectangular,oval, elliptical, or any combination of the same. In certainembodiments, the spacers have pillar shape, have a substantially flattop surface, and have substantially uniform cross-section, wherein, foreach spacer, the ratio of the lateral dimension of the spacer to itsheight is at least 1. In certain embodiments, the inter spacer distanceis periodic. In certain embodiments, the spacers have a filling factorof 1% or higher, wherein the filling factor is the ratio of the spacercontact area to the total plate area. In certain embodiments, theYoung's modulus of the spacers times the filling factor of the spacersis equal or larger than 20 MPa, wherein the filling factor is the ratioof the spacer contact area to the total plate area. In certainembodiments, the spacing between the two plates at the closedconfiguration is in less 200 um. In certain embodiments, the spacingbetween the two plates at the closed configuration is a value selectedfrom between 1.8 um and 3.5 um. In certain embodiments, the spacing arefixed on a plate by directly embossing the plate or injection molding ofthe plate. In certain embodiments, the materials of the plate and thespacers are selected from polystyrene, PMMA, PC, COC, COP, or anotherplastic. In certain embodiments, the spacers have a pillar shape, andthe sidewall corners of the spacers have a round shape with a radius ofcurvature at least 1 μm. In certain embodiments, the spacers have adensity of at least 1000/mm². In certain embodiments, at least one ofthe plates is transparent. In certain embodiments, the mold used to makethe spacers is fabricated by a mold containing features that arefabricated by either (a) directly reactive ion etching or ion beametched or (b) by a duplication or multiple duplication of the featuresthat are reactive ion etched or ion beam etched.

In certain embodiments, the spacers are configured, such that thefilling factor is in the range of 1% to 5%. In certain embodiments, thesurface variation is relative to the spacer height and the ratio of thepillar flat top surface variation to the spacer height is less than0.5%, 1%, 3%, 5%, 7%, 10%, 15%, 20%, 30%, 40%, or in a range between anytwo of the values. A preferred flat pillar top smoothness has a ratio ofthe pillar flat top surface variation to the spacer height is less than2%, 5%, or 10%. In certain embodiments, the spacers are configured, suchthat the filling factor is in the range of 1% to 5%. In certainembodiments, the spacers are configured, such that the filling factor isin the range of 5% to 10%. In certain embodiments, the spacers areconfigured, such that the filling factor is in the range of 10% to 20%.In certain embodiments, the spacers are configured, such that thefilling factor is in the range of 20% to 30%. In certain embodiments,the spacers are configured, such that the filling factor is 5%, 10%,20%, 30%, 40%, 50%, or in a range of any two of the values. In certainembodiments, the spacers are configured, such that the filling factor is50%, 60%, 70%, 80%, or in a range of any two of the values.

In certain embodiments, the spacers are configured, such that thefilling factor multiplies the Young's modulus of the spacer is in therange of 2 MPa and 10 MPa. In certain embodiments, the spacers areconfigured, such that the filling factor multiplies the Young's modulusof the spacer is in the range of 10 MPa and 20 MPa. In certainembodiments, the spacers are configured, such that the filling factormultiplies the Young's modulus of the spacer is in the range of 20 MPaand 40 MPa. In certain embodiments, the spacers are configured, suchthat the filling factor multiplies the Young's modulus of the spacer isin the range of 40 MPa and 80 MPa. In certain embodiments, the spacersare configured, such that the filling factor multiplies the Young'smodulus of the spacer is in the range of 80 MPa and 120 MPa. In certainembodiments, the spacers are configured, such that the filling factormultiplies the Young's modulus of the spacer is in the range of 120 MPato 150 MPa.

In certain embodiments, the device further comprises a dry reagentcoated on one or both plates. In certain embodiments, the device furthercomprises, on one or both plates, a dry binding site that has apredetermined area, wherein the dry binding site binds to andimmobilizes an analyte in the sample. In certain embodiments, the devicefurther comprises, on one or both plates, a releasable dry reagent and arelease time control material that delays the time that the releasabledry regent is released into the sample. In certain embodiments, therelease time control material delays the time that the dry regent startsis released into the sample by at least 3 seconds. In certainembodiments, the regent comprises anticoagulant and/or stainingreagent(s). In certain embodiments, the reagent comprises cell lysingreagent(s). In certain embodiments, the device further comprises, on oneor both plates, one or a plurality of dry binding sites and/or one or aplurality of reagent sites. In certain embodiments, the analytecomprises a molecule (e.g., a protein, peptides, DNA, RNA, nucleic acid,or other molecule), cells, tissues, viruses, and nanoparticles withdifferent shapes. In certain embodiments, the analyte comprises whiteblood cells, red blood cells and platelets. In certain embodiments, theanalyte is stained.

In certain embodiments, the spacers regulating the layer of uniformthickness have a filling factor of at least 1%, wherein the fillingfactor is the ratio of the spacer area in contact with the layer ofuniform thickness to the total plate area in contact with the layer ofuniform thickness. In certain embodiments, for spacers regulating thelayer of uniform thickness, the Young's modulus of the spacers times thefilling factor of the spacers is equal or larger than 10 MPa, whereinthe filling factor is the ratio of the spacer area in contact with thelayer of uniform thickness to the total plate area in contact with thelayer of uniform thickness. In certain embodiments, for a flexibleplate, the thickness of the flexible plate times the Young's modulus ofthe flexible plate is in the range 60 to 750 GPa-um. In certainembodiments, for a flexible plate, the fourth power of theinter-spacer-distance (ISD) divided by the thickness of the flexibleplate (h) and the Young's modulus (E) of the flexible plate, ISD⁴/(hE),is equal to or less than 10⁶ um³/GPa.

In certain embodiments, one or both plates comprises a location marker,either on a surface of or inside the plate, that provide information ofa location of the plate. In certain embodiments, one or both platescomprises a scale marker, either on a surface of or inside the plate,that provide information of a lateral dimension of a structure of thesample and/or the plate. In certain embodiments, one or both platescomprises an imaging marker, either on surface of or inside the plate,that assists an imaging of the sample. In certain embodiments, thespacers functions as a location marker, a scale marker, an imagingmarker, or any combination of thereof.

In certain embodiments, the average thickness of the layer of uniformthickness is about equal to a minimum dimension of an analyte in thesample. In certain embodiments, the inter-spacer distance is in therange of 7 μm to 50 μm. In certain embodiments, the inter-spacerdistance is in the range of 50 μm to 120 μm. In certain embodiments, theinter-spacer distance is in the range of 120 μm to 200 μm (micron). Incertain embodiments, the inter-spacer distance is substantiallyperiodic. In certain embodiments, the spacers are pillars with across-sectional shape selected from round, polygonal, circular, square,rectangular, oval, elliptical, or any combination of the same.

In certain embodiments, the spacers have a pillar shape and have asubstantially flat top surface, wherein, for each spacer, the ratio ofthe lateral dimension of the spacer to its height is at least 1. Incertain embodiments, each spacer has the ratio of the lateral dimensionof the spacer to its height is at least 1. In certain embodiments, theminimum lateral dimension of spacer is less than or substantially equalto the minimum dimension of an analyte in the sample. In certainembodiments, the minimum lateral dimension of spacer is in the range of0.5 um to 100 um. In certain embodiments, the minimum lateral dimensionof spacer is in the range of 0.5 um to 10 um.

In certain embodiments, the sample is blood. In certain embodiments, thesample is whole blood without dilution by liquid. In certainembodiments, the sample is a biological sample selected from amnioticfluid, aqueous humour, vitreous humour, blood (e.g., whole blood,fractionated blood, plasma or serum), breast milk, cerebrospinal fluid(CSF), cerumen (earwax), chyle, chime, endolymph, perilymph, feces,breath, gastric acid, gastric juice, lymph, mucus (including nasaldrainage and phlegm), pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, exhaled breath condensates, sebum, semen,sputum, sweat, synovial fluid, tears, vomit, and urine. In certainembodiments, the sample is a biological sample, an environmental sample,a chemical sample, or clinical sample.

In certain embodiments, the spacers have a pillar shape, and thesidewall corners of the spacers have a round shape with a radius ofcurvature at least 1 μm. In certain embodiments, the spacers have adensity of at least 100/mm². In certain embodiments, the spacers have adensity of at least 1000/mm². In certain embodiments, at least one ofthe plates is transparent. In certain embodiments, at least one of theplates is made from a flexible polymer. In certain embodiments, for apressure that compresses the plates, the spacers are not compressibleand/or, independently, only one of the plates is flexible. In certainembodiments, the flexible plate has a thickness in the range of 10 um to200 um. In certain embodiments, the variation is less than 30%. Incertain embodiments, the variation is less than 10%. In certainembodiments, the variation is less than 5%.

In certain embodiments, the first and second plates are connected andare configured to be changed from the open configuration to the closedconfiguration by folding the plates. In certain embodiments, the firstand second plates are connected by a hinge and are configured to bechanged from the open configuration to the closed configuration byfolding the plates along the hinge. In certain embodiments, the firstand second plates are connected by a hinge that is a separate materialto the plates, and are configured to be changed from the openconfiguration to the closed configuration by folding the plates alongthe hinge. In certain embodiments, the first and second plates are madein a single piece of material and are configured to be changed from theopen configuration to the closed configuration by folding the plates. Incertain embodiments, the layer of uniform thickness sample is uniformover a lateral area that is at least 1 mm².

In certain embodiments, the device is configured to analyze the samplein 60 seconds or less. In certain embodiments, at the closedconfiguration, the final sample thickness device is configured toanalyze the sample in 60 seconds or less. In certain embodiments, at theclosed configuration, the final sample thickness device is configured toanalyze the sample in 10 seconds or less.

In certain embodiments, the dry binding site comprises a capture agent.In certain embodiments, the dry binding site comprises an antibody ornucleic acid. In certain embodiments, the releasable dry reagent is alabeled reagent. In certain embodiments, the releasable dry reagent is afluorescently-labeled reagent. In certain embodiments, the releasabledry reagent is a fluorescently-labeled antibody. In certain embodiments,the releasable dry reagent is a cell stain. In certain embodiments, thereleasable dry reagent is a cell lysing.

In certain embodiments, the detector is an optical detector that detectsan optical signal. In certain embodiments, the detector is an electricdetector that detect electrical signal. In certain embodiments, thespacing are fixed on a plate by directly embossing the plate orinjection molding of the plate. In certain embodiments, the materials ofthe plate and the spacers are selected from polystyrene, PMMA, PC, COC,COP, or another plastic.

In certain embodiments of the present disclosure, a system for rapidlyanalyzing a sample using a mobile phone can comprise a device of anyprior embodiment. In certain embodiments of the present disclosure, asystem for rapidly analyzing a sample using a mobile phone can comprisea mobile communication device. In certain embodiments, the mobilecommunication device can comprise one or a plurality of cameras for thedetecting and/or imaging the sample. In certain embodiments, the mobilecommunication device can comprise electronics, signal processors,hardware and software for receiving and/or processing the detectedsignal and/or the image of the sample and for remote communication. Incertain embodiments, the mobile communication device can comprise alight source from either the mobile communication device or an externalsource. In same embodiments, the detector in the devices or methods ofany prior embodiment is provided by the mobile communication device, anddetects an analyte in the sample at the closed configuration.

In certain embodiments, one of the plates has a binding site that bindsan analyte, wherein at least part of the uniform sample thickness layeris over the binding site, and is substantially less than the averagelateral linear dimension of the binding site. In certain embodiments,any system of the present disclosure can comprise a housing configuredto hold the sample and to be mounted to the mobile communication device.In certain embodiments, the housing comprises optics for facilitatingthe imaging and/or signal processing of the sample by the mobilecommunication device, and a mount configured to hold the optics on themobile communication device. In certain embodiments, an element of theoptics in the housing is movable relative to the housing. In certainembodiments, the mobile communication device is configured tocommunicate test results to a medical professional, a medical facilityor an insurance company. In certain embodiments, the mobilecommunication device is further configured to communicate information onthe test and the subject with the medical professional, medical facilityor insurance company. In certain embodiments, the mobile communicationdevice is further configured to communicate information of the test to acloud network, and the cloud network process the information to refinethe test results. In certain embodiments, the mobile communicationdevice is further configured to communicate information of the test andthe subject to a cloud network, the cloud network process theinformation to refine the test results, and the refined test resultswill send back the subject. In certain embodiments, the mobilecommunication device is configured to receive a prescription, diagnosisor a recommendation from a medical professional. In certain embodiments,the mobile communication device is configured with hardware and softwareto capture an image of the sample. In certain embodiments, the mobilecommunication device is configured with hardware and software to analyzea test location and a control location in in image. In certainembodiments, the mobile communication device is configured with hardwareand software to compare a value obtained from analysis of the testlocation to a threshold value that characterizes the rapid diagnostictest.

In certain embodiments of the present disclosure, at least one of theplates comprises a storage site in which assay reagents are stored. Incertain embodiments, at least one of the cameras reads a signal from thedevice. In certain embodiments, the mobile communication devicecommunicates with the remote location via a wifi or cellular network. Incertain embodiments, the mobile communication device is a mobile phone.

In certain embodiments of the present disclosure, a method for rapidlyanalyzing an analyte in a sample using a mobile phone can comprisedepositing a sample on the device of any prior system embodiment. Incertain embodiments of the present disclosure, a method for rapidlyanalyzing an analyte in a sample using a mobile phone can compriseassaying an analyte in the sample deposited on the device to generate aresult. In certain embodiments of the present disclosure, a method forrapidly analyzing an analyte in a sample using a mobile phone cancomprise communicating the result from the mobile communication deviceto a location remote from the mobile communication device.

In certain embodiments, the analyte comprises a molecule (e.g., aprotein, peptides, DNA, RNA, nucleic acid, or other molecule), cells,tissues, viruses, and nanoparticles with different shapes. In certainembodiments, the analyte comprises white blood cell, red blood cell andplatelets. In certain embodiments, the assaying comprises performing awhite blood cells differential assay. In certain embodiments, a methodof the present disclosure can comprise analyzing the results at theremote location to provide an analyzed result. In certain embodiments, amethod of the present disclosure can comprise communicating the analyzedresult from the remote location to the mobile communication device. Incertain embodiments, the analysis is done by a medical professional at aremote location. In certain embodiments, the mobile communication devicereceives a prescription, diagnosis or a recommendation from a medicalprofessional at a remote location.

In certain embodiments, the sample is a bodily fluid. In certainembodiments, the bodily fluid is blood, saliva or urine. In certainembodiments, the sample is whole blood without dilution by a liquid. Incertain embodiments, the assaying step comprises detecting an analyte inthe sample. In certain embodiments, the analyte is a biomarker. Incertain embodiments, the analyte is a protein, nucleic acid, cell, ormetabolite. In certain embodiments, the method comprises counting thenumber of red blood cells. In certain embodiments, the method comprisescounting the number of white blood cells. In certain embodiments, themethod comprises staining the cells in the sample and counting thenumber of neutrophils, lymphocytes, monocytes, eosinophils andbasophils. In certain embodiments, the assay done in step (b) is abinding assay or a biochemical assay.

In certain embodiments of the present disclosure, a method for analyzinga sample can comprise obtaining a device of any prior device embodiment.In certain embodiments of the present disclosure, a method for analyzinga sample can comprise depositing the sample onto one or both pates ofthe device. In certain embodiments of the present disclosure, a methodfor analyzing a sample can comprise placing the plates in a closedconfiguration and applying an external force over at least part of theplates. In certain embodiments of the present disclosure, a method foranalyzing a sample can comprise analyzing the layer of uniform thicknesswhile the plates are the closed configuration.

In certain embodiments, the first plate further comprises, on itssurface, a first predetermined assay site and a second predeterminedassay site, wherein the distance between the edges of the assay site issubstantially larger than the thickness of the uniform thickness layerwhen the plates are in the closed position, wherein at least a part ofthe uniform thickness layer is over the predetermined assay sites, andwherein the sample has one or a plurality of analytes that are capableof diffusing in the sample. In certain embodiments, the first plate has,on its surface, at least three analyte assay sites, and the distancebetween the edges of any two neighboring assay sites is substantiallylarger than the thickness of the uniform thickness layer when the platesare in the closed position, wherein at least a part of the uniformthickness layer is over the assay sites, and wherein the sample has oneor a plurality of analytes that are capable of diffusing in the sample.In certain embodiments, the first plate has, on its surface, at leasttwo neighboring analyte assay sites that are not separated by a distancethat is substantially larger than the thickness of the uniform thicknesslayer when the plates are in the closed position, wherein at least apart of the uniform thickness layer is over the assay sites, and whereinthe sample has one or a plurality of analytes that are capable ofdiffusing in the sample. In certain embodiments, the analyte assay areais between a pair of electrodes. In certain embodiments, the assay areais defined by a patch of dried reagent. In certain embodiments, theassay area binds to and immobilizes the analyte. In certain embodiments,the assay area is defined by a patch of binding reagent that, uponcontacting the sample, dissolves into the sample, diffuses in thesample, and binds to the analyte. In certain embodiments, theinter-spacer distance is in the range of 14 μm to 200 μm. In certainembodiments, the inter-spacer distance is in the range of 7 μm to 20 μm.In certain embodiments, the spacers are pillars with a cross-sectionalshape selected from round, polygonal, circular, square, rectangular,oval, elliptical, or any combination of the same. In certainembodiments, the spacers have a pillar shape and have a substantiallyflat top surface, wherein, for each spacer, the ratio of the lateraldimension of the spacer to its height is at least 1. In certainembodiments, the spacers have a pillar shape, and the sidewall cornersof the spacers have a round shape with a radius of curvature at least 1μm. In certain embodiments, the spacers have a density of at least1000/mm². In certain embodiments, at least one of the plates istransparent. In certain embodiments, at least one of the plates is madefrom a flexible polymer. In certain embodiments, only one of the platesis flexible. In certain embodiments, the area-determination device is acamera. In certain embodiments, an area in the sample contact area of aplate, wherein the area is less than 1/100, 1/20, 1/10, 1/6, 1/5, 1/4,1/3, 1/2, 2/3 of the sample contact area, or in a range between any ofthe two values. In certain embodiments, the area-determination devicecomprises a camera and an area in the sample contact area of a plate,wherein the area is in contact with the sample.

In certain embodiments, the deformable sample comprises a liquid sample.In certain embodiments, the imprecision force has a variation at least30% of the total force that actually is applied. In certain embodiments,the imprecision force has a variation at least 20%, 30%, 40%, 50%, 60,70%, 80%, 90% 100%, 150%, 200%, 300%, 500%, or in a range of any twovalues, of the total force that actually is applied. In certainembodiments, the spacers have a flat top. In certain embodiments, thedevice is further configured to have, after the pressing force isremoved, a sample thickness that is substantially the same in thicknessand uniformity as that when the force is applied. In certainembodiments, the imprecise force is provided by human hand. In certainembodiments, the inter spacer distance is substantially constant. Incertain embodiments, the inter spacer distance is substantially periodicin the area of the uniform sample thickness area. In certainembodiments, the multiplication product of the filling factor and theYoung's modulus of the spacer is 2 MPa or larger. In certainembodiments, the force is applied by hand directly or indirectly. Incertain embodiments, the force applied is in the range of 1 N to 20 N.In certain embodiments, the force applied is in the range of 20 N to 200N. In certain embodiments, the highly uniform layer has a thickness thatvaries by less than 15%, 10%, or 5% of an average thickness. In certainembodiments, the imprecise force is applied by pinching the devicebetween a thumb and forefinger. In certain embodiments, thepredetermined sample thickness is larger than the spacer height. Incertain embodiments, the device holds itself in the closed configurationafter the pressing force has been removed. In certain embodiments, theuniform thickness sample layer area is larger than that area upon whichthe pressing force is applied. In certain embodiments, the spacers donot significantly deform during application of the pressing force. Incertain embodiments, the pressing force is not predetermined beforehandand is not measured. In certain embodiments, the fluidic sample isreplaced by a deformable sample and the embodiments for making at leasta part of the fluidic sample into a uniform thickness layer can make atleast a part of the deformable sample into a uniform thickness layer. Incertain embodiments, the inter spacer distance is periodic. In certainembodiments, the spacers have a flat top. In certain embodiments, theinter spacer distance is at least two times large than the size of thetargeted analyte in the sample.

Manufacturing of Q-Card

In certain embodiments of the present disclosure, a Q-Card can comprisea first plate. In certain embodiments of the present disclosure, aQ-Card can comprise a second plate. In certain embodiments of thepresent disclosure, a Q-Card can comprise a hinge. In certainembodiments, the first plate, that is about 200 nm to 1500 nm thick,comprises, on its inner surface, (a) a sample contact area forcontacting a sample, and (b) a sample overflow dam that surrounds thesample contact area is configured to present a sample flow outside ofthe dam. In certain embodiments, the second plate is 10 um to 250 umthick and comprises, on its inner surface, (a) a sample contact area forcontacting a sample, and (b) spacers on the sample contact area. Incertain embodiments, the hinge that connect the first and the secondplates. In certain embodiments, the first and second plate are movablerelative to each other around the axis of the hinge.

In certain embodiments of the present disclosure, an embodiment of theQ-Card can comprise a first plate. In certain embodiments of the presentdisclosure, an embodiment of the Q-Card can comprise a second plate. Incertain embodiments of the present disclosure, an embodiment of theQ-Card can comprise a hinge. In certain embodiments, the first plate,that is about 200 nm to 1500 nm thick, comprises, on its inner surface,(a) a sample contact area for contacting a sample, (b) a sample overflowdam that surrounds the sample contact area is configured to present asample flow outside of the dam, and (c) spacers on the sample contactarea. In certain embodiments, the second plate, that is 10 um to 250 umthick, comprises, on its inner surface, a sample contact area forcontacting a sample. In certain embodiments, the hinge connects thefirst and the second plates. In certain embodiments, the first andsecond plate are movable relative to each other around the axis of thehinge.

In certain embodiments of the present disclosure, an embodiment of theQ-Card can comprise a first plate. In certain embodiments of the presentdisclosure, an embodiment of the Q-Card can comprise a second plate. Incertain embodiments of the present disclosure, an embodiment of theQ-Card can comprise a hinge. In certain embodiments, the first plate,that is about 200 nm to 1500 nm thick, comprises, on its inner surface,(a) a sample contact area for contacting a sample, and (b) spacers onthe sample contact area. In certain embodiments, the second plate, thatis 10 um to 250 um thick, comprises, on its inner surface, (a) a samplecontact area for contacting a sample, and (b) a sample overflow darnthat surrounds the sample contact area is configured to present a sampleflow outside of the darn. In certain embodiments, the hinge connects thefirst and the second plates. In certain embodiments, the first andsecond plate are movable relative to each other around the axis of thehinge.

In certain embodiments of the present disclosure, an embodiment of theQ-Card can comprise a first plate. In certain embodiments of the presentdisclosure, an embodiment of the Q-Card can comprise a second plate. Incertain embodiments of the present disclosure, an embodiment of theQ-Card can comprise a hinge. In certain embodiments, the first plate,that is about 200 nm to 1500 nm thick, comprises, on its inner surface,a sample contact area for contacting a sample. In certain embodiments,the second plate, that is 10 um to 250 um thick, comprises, on its innersurface, (a) a sample contact area for contacting a sample, (b) a sampleoverflow darn that surrounds the sample contact area is configured topresent a sample flow outside of the darn, and (c) spacers on the samplecontact area. In certain embodiments, the hinge connects the first andthe second plates. In certain embodiments, the first and second plateare movable relative to each other around the axis of the hinge.

In certain embodiments of the present disclosure, a method forfabricating any Q-Card of the present disclosure can comprise injectionmolding of the first plate. In certain embodiments of the presentdisclosure, a method for fabricating any Q-Card of the presentdisclosure can comprise nanoimprinting or extrusion printing of thesecond plate.

In certain embodiments of the present disclosure, a method forfabricating any Q-Card of the present disclosure can comprise Lasercutting the first plate. In certain embodiments of the presentdisclosure, a method for fabricating any Q-Card of the presentdisclosure can comprise nanoimprinting or extrusion printing of thesecond plate.

In certain embodiments of the present disclosure, a method forfabricating any Q-Card of the present disclosure can comprise injectionmolding and laser cutting the first plate. In certain embodiments of thepresent disclosure, a method for fabricating any Q-Card of the presentdisclosure can comprise nanoimprinting or extrusion printing of thesecond plate.

In certain embodiments of the present disclosure, a method forfabricating any Q-Card of the present disclosure can comprisenanoimprinting or extrusion printing to fabricated both the first andthe second plate.

In certain embodiments of the present disclosure, a method forfabricating any Q-Card of the present disclosure can comprisefabricating the first plate or the second plate, using injectionmolding, laser cutting the first plate, nanoimprinting, extrusionprinting, or a combination of thereof.

In certain embodiments of the present disclosure, a method forfabricating any Q-Card of the present disclosure can comprise a step ofattaching the hinge on the first and the second plates after thefabrication of the first and second plates.

Additional Examples and Definitions (1) Definitions

The terms used in describing the devices/apparatus, systems, and methodsherein disclosed are defined in the current application, or in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

(2) Sample

The devices/apparatus, systems, and methods herein disclosed can beapplied to manipulation and detection of various types of samples. Thesamples are herein disclosed, listed, described, and/or summarized inPCT Application (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

The devices, apparatus, systems, and methods herein disclosed can beused for samples such as but not limited to diagnostic samples, clinicalsamples, environmental samples and foodstuff samples. The types ofsample include but are not limited to the samples listed, describedand/or summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, and are hereby incorporated byreference by their entireties.

For example, in some embodiments, the devices, apparatus, systems, andmethods herein disclosed are used for a sample that includes cells,tissues, bodily fluids and/or a mixture thereof. In some embodiments,the sample comprises a human body fluid. In some embodiments, the samplecomprises at least one of cells, tissues, bodily fluids, stool, amnioticfluid, aqueous humour, vitreous humour, blood, whole blood, fractionatedblood, plasma, serum, breast milk, cerebrospinal fluid, cerumen, chyle,chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph,mucus, nasal drainage, phlegm, pericardial fluid, peritoneal fluid,pleural fluid, pus, rheum, saliva, sebum, semen, sputum, sweat, synovialfluid, tears, vomit, urine, and exhaled breath condensate.

In some embodiments, the devices, apparatus, systems, and methods hereindisclosed are used for an environmental sample that is obtained from anysuitable source, such as but not limited to: river, lake, pond, ocean,glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinkingwater, etc.; solid samples from soil, compost, sand, rocks, concrete,wood, brick, sewage, etc.; and gaseous samples from the air, underwaterheat vents, industrial exhaust, vehicular exhaust, etc. In certainembodiments, the environmental sample is fresh from the source; incertain embodiments, the environmental sample is processed. For example,samples that are not in liquid form are converted to liquid form beforethe subject devices, apparatus, systems, and methods are applied.

In some embodiments, the devices, apparatus, systems, and methods hereindisclosed are used for a foodstuff sample, which is suitable or has thepotential to become suitable for animal consumption, e.g., humanconsumption. In some embodiments, a foodstuff sample includes rawingredients, cooked or processed food, plant and animal sources of food,preprocessed food as well as partially or fully processed food, etc. Incertain embodiments, samples that are not in liquid form are convertedto liquid form before the subject devices, apparatus, systems, andmethods are applied.

The subject devices, apparatus, systems, and methods can be used toanalyze any volume of the sample. Examples of the volumes include, butare not limited to, about 10 mL or less, 5 mL or less, 3 mL or less, 1microliter (μL, also “uL” herein) or less, 500 μL or less, 300 μL orless, 250 μL or less, 200 μL or less, 170 μL or less, 150 μL or less,125 μL or less, 100 μL or less, 75 μL or less, 50 μL or less, 25 μL orless, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL orless, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less,0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 pL or less, 1pL or less, or a range between any two of the values.

In some embodiments, the volume of the sample includes, but is notlimited to, about 100 μL or less, 75 μL or less, 50 μL or less, 25 μL orless, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL orless, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less,0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 pL or less, 1pL or less, or a range between any two of the values. In someembodiments, the volume of the sample includes, but is not limitedto,about 10 μL or less, 5 μL or less, 3 μL or less, 1 μL or less, 0.5 μLor less, 0.1 μL or less, 0.05 μL or less, 0.001 μL or less, 0.0005 μL orless, 0.0001 μL or less, 10 pL or less, 1 pL or less, or a range betweenany two of the values.

In some embodiments, the amount of the sample is about a drop of liquid.In certain embodiments, the amount of sample is the amount collectedfrom a pricked finger or fingerstick. In certain embodiments, the amountof sample is the amount collected from a microneedle, micropipette or avenous draw.

In certain embodiments, the sample holder is configured to hold afluidic sample. In certain embodiments, the sample holder is configuredto compress at least part of the fluidic sample into a thin layer. Incertain embodiments, the sample holder comprises structures that areconfigured to heat and/or cool the sample. In certain embodiments, theheating source provides electromagnetic waves that can be absorbed bycertain structures in the sample holder to change the temperature of thesample. In certain embodiments, the signal sensor is configured todetect and/or measure a signal from the sample. In certain embodiments,the signal sensor is configured to detect and/or measure an analyte inthe sample. In certain embodiments, the heat sink is configured toabsorb heat from the sample holder and/or the heating source. In certainembodiments, the heat sink comprises a chamber that at least partlyenclose the sample holder.

(3) Q-Card, Spacers and Uniform Sample thickness

The devices/apparatus, systems, and methods herein disclosed can includeor use Q-cards, spacers, and uniform sample thickness embodiments forsample detection, analysis, and quantification. In some embodiments, theQ-card comprises spacers, which help to render at least part of thesample into a layer of high uniformity. The structure, material,function, variation and dimension of the spacers, as well as theuniformity of the spacers and the sample layer, are herein disclosed,listed, described, and/or summarized in PCT Application (designatingU.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which wererespectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S. ProvisionalApplication No. 62/456,065, which was filed on Feb. 7, 2017, U.S.Provisional Application No. 62/456,287, which was filed on Feb. 8, 2017,and U.S. Provisional Application No. 62/456,504, which was filed on Feb.8, 2017, all of which applications are incorporated herein in theirentireties for all purposes.

The term “open configuration” of the two plates in a QMAX process meansa configuration in which the two plates are either partially orcompletely separated apart and the spacing between the plates is notregulated by the spacers

The term “closed configuration” of the two plates in a QMAX processmeans a configuration in which the plates are facing each other, thespacers and a relevant volume of the sample are between the plates, therelevant spacing between the plates, and thus the thickness of therelevant volume of the sample, is regulated by the plates and thespacers, wherein the relevant volume is at least a portion of an entirevolume of the sample.

The term “a sample thickness is regulated by the plate and the spacers”in a QMAX process means that for a give condition of the plates, thesample, the spacer, and the plate compressing method, the thickness ofat least a port of the sample at the closed configuration of the platescan be predetermined from the properties of the spacers and the plate.

The term “inner surface” or “sample surface” of a plate in a QMAX cardrefers to the surface of the plate that touches the sample, while theother surface (that does not touch the sample) of the plate is termed“outer surface”.

The term “height” or “thickness” of an object in a QMAX process refersto, unless specifically stated, the dimension of the object that is inthe direction normal to a surface of the plate. For example, spacerheight is the dimension of the spacer in the direction normal to asurface of the plate, and the spacer height and the spacer thicknessmeans the same thing.

The term “area” of an object in a QMAX process refers to, unlessspecifically stated, the area of the object that is parallel to asurface of the plate. For example, spacer area is the area of the spacerthat is parallel to a surface of the plate.

The term of QMAX card refers the device that perform a QMAX (e.g. CROF)process on a sample, and have or not have a hinge that connect the twoplates.

The term “QMAX card with a hinge and “QMAX card” are interchangeable.

The term “angle self-maintain”, “angle self-maintaining”, or “rotationangle self-maintaining” refers to the property of the hinge, whichsubstantially maintains an angle between the two plates, after anexternal force that moves the plates from an initial angle into theangle is removed from the plates.

In using QMAX card, the two plates need to be open first for sampledeposition. However, in some embodiments, the QMAX card from a packagehas the two plates are in contact each other (e.g. a close position),and to separate them is challenges, since one or both plates are verything. To facilitate an opening of the QMAX card, opening notch ornotches are created at the edges or corners of the first plate or bothplaces, and, at the close position of the plates, a part of the secondplate placed over the opening notch, hence in the notch of the firstplate, the second plate can be lifted open without a blocking of thefirst plate.

In the QMAX assay platform, a QMAX card uses two plates to manipulatethe shape of a sample into a thin layer (e.g. by compressing). Incertain embodiments, the plate manipulation needs to change the relativeposition (termed: plate configuration) of the two plates several timesby human hands or other external forces. There is a need to design theQMAX card to make the hand operation easy and fast.

In QMAX assays, one of the plate configurations is an openconfiguration, wherein the two plates are completely or partiallyseparated (the spacing between the plates is not controlled by spacers)and a sample can be deposited. Another configuration is a closedconfiguration, wherein at least part of the sample deposited in the openconfiguration is compressed by the two plates into a layer of highlyuniform thickness, the uniform thickness of the layer is confined by theinner surfaces of the plates and is regulated by the plates and thespacers. In some embodiments, the average spacing between the two platesis more than 300 um.

In a QMAX assay operation, an operator needs to first make the twoplates to be in an open configuration ready for sample deposition, thendeposit a sample on one or both of the plates, and finally close theplates into a close position. In certain embodiments, the two plates ofa QMAX card are initially on top of each other and need to be separatedto get into an open configuration for sample deposition. When one of theplate is a thin plastic film (175 um thick PMA), such separation can bedifficult to perform by hand. The present invention intends to providethe devices and methods that make the operation of certain assays, suchas the QMAX card assay, easy and fast.

In some embodiments, the QMAX device comprises a hinge that connect twoor more plates together, so that the plates can open and close in asimilar fashion as a book. In some embodiments, the material of thehinge is such that the hinge can self-maintain the angle between theplates after adjustment. In some embodiments, the hinge is configured tomaintain the QMAX card in the closed configuration, such that the entireQMAX card can be slide in and slide out a card slot without causingaccidental separation of the two plates. In some embodiments, the QMAXdevice comprises one or more hinges that can control the rotation ofmore than two plates.

In some embodiments, the hinge is made from a metallic material that isselected from a group consisting of gold, silver, copper, aluminum,iron, tin, platinum, nickel, cobalt, alloys, or any combination ofthereof. In some embodiments, the hinge comprises a single layer, whichis made from a polymer material, such as but not limited to plastics.The polymer material is selected from the group consisting of acrylatepolymers, vinyl polymers, olefin polymers, cellulosic polymers,noncellulosic polymers, polyester polymers, Nylon, cyclic olefincopolymer (COC), poly(methyl methacrylate) (PMMB), polycarbonate (PC),cyclic olefin polymer (COP), liquid crystalline polymer (LCP), polyimide(PB), polyethylene (PE), polyimide (PI), polypropylene (PP),poly(phenylene ether) (PPE), polystyrene (PS), polyoxymethylene (POM),polyether ether ketone (PEEK), polyether sulfone (PES), poly(ethylenephthalate) (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride(PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT),fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFB),polydimethylsiloxane (PDMS), rubbers, or any combinations of thereof. Insome embodiments, the polymer material is selected from polystyrene,PMMB, PC, COC, COP, other plastic, or any combination of thereof.

In essence, the term “spacers” or “stoppers” refers to, unless statedotherwise, the mechanical objects that set, when being placed betweentwo plates, a limit on the minimum spacing between the two plates thatcan be reached when compressing the two plates together. Namely, in thecompressing, the spacers will stop the relative movement of the twoplates to prevent the plate spacing becoming less than a preset (i.e.predetermined) value.

The term “a spacer has a predetermined height” and “spacers have apredetermined inter-spacer distance” means, respectively, that the valueof the spacer height and the inter spacer distance is known prior to aQMAX process. It is not predetermined, if the value of the spacer heightand the inter-spacer distance is not known prior to a QMAX process. Forexample, in the case that beads are sprayed on a plate as spacers, wherebeads are landed at random locations of the plate, the inter-spacerdistance is not predetermined. Another example of not predeterminedinter spacer distance is that the spacers moves during a QMAX processes.

The term “a spacer is fixed on its respective plate” in a QMAX processmeans that the spacer is attached to a location of a plate and theattachment to that location is maintained during a QMAX (i.e. thelocation of the spacer on respective plate does not change) process. Anexample of “a spacer is fixed with its respective plate” is that aspacer is monolithically made of one piece of material of the plate, andthe location of the spacer relative to the plate surface does not changeduring the QMAX process. An example of “a spacer is not fixed with itsrespective plate” is that a spacer is glued to a plate by an adhesive,but during a use of the plate, during the QMAX process, the adhesivecannot hold the spacer at its original location on the plate surface andthe spacer moves away from its original location on the plate surface.

In some embodiments, human hands can be used to press the plates into aclosed configuration; In some embodiments, human hands can be used topress the sample into a thin layer. The manners in which hand pressingis employed are described and/or summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 filed on Aug. 10, 2016 andPCT/US0216/051775 filed on Sep. 14, 2016, and in U.S. ProvisionalApplication Nos. 62/431,639 filed on Dec. 9, 2016, 62/456,287 filed onFeb. 8, 2017, 62/456,065 filed on Feb. 7, 2017, 62/456,504 filed on Feb.8, 2017, and 62/460,062 filed on Feb. 16, 2017, which are all herebyincorporated by reference by their entireties.

In some embodiments, human hand can be used to manipulate or handle theplates of the QMAX device. In certain embodiments, the human hand can beused to apply an imprecise force to compress the plates from an openconfiguration to a closed configuration. In certain embodiments, thehuman hand can be used to apply an imprecise force to achieve high levelof uniformity in the thickness of the sample (e.g. less than 5%, 10%,15%, or 20% variability).

(4) Hinges, Opening Notches, Recessed Edge and Sliders

The devices/apparatus, systems, and methods herein disclosed can includeor use Q-cards for sample detection, analysis, and quantification. Insome embodiments, the Q-card comprises hinges, notches, recesses, andsliders, which help to facilitate the manipulation of the Q card and themeasurement of the samples. The structure, material, function, variationand dimension of the hinges, notches, recesses, and sliders are hereindisclosed, listed, described, and/or summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, whichwere respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.Provisional Application No. 62/431,639, which was filed on Dec. 9, 2016,U.S. Provisional Application No. 62/456,065, which was filed on Feb. 7,2017, U.S. Provisional Application Nos. 62/456,287 and 62/456,504, whichwas filed on Feb. 8, 2017, and U.S. Provisional Application No.62/539,660, which was filed on Aug. 1, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

In some embodiments, the QMAX device comprises opening mechanisms suchas but not limited to notches on plate edges or strips attached to theplates, making is easier for a user to manipulate the positioning of theplates, such as but not limited to separating the plates of by hand.

In some embodiments, the QMAX device comprises trenches on one or bothof the plates. In certain embodiments, the trenches limit the flow ofthe sample on the plate.

(5) Q-Card and Adaptor

The devices/apparatus, systems, and methods herein disclosed can includeor use Q-cards for sample detection, analysis, and quantification. Insome embodiments, the Q-card is used together with an adaptor that isconfigured to accommodate the Q-card and connect to a mobile device sothat the sample in the Q-card can be imaged, analyzed, and/or measuredby the mobile device. The structure, material, function, variation,dimension and connection of the Q-card, the adaptor, and the mobile areherein disclosed, listed, described, and/or summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application Nos. 62/456,287 and62/456,590, which were filed on Feb. 8, 2017, U.S. ProvisionalApplication No. 62/456,504, which was filed on Feb. 8, 2017, U.S.Provisional Application No. 62/459,544, which was filed on Feb. 15,2017, and U.S. Provisional Application No. 62/460,075 and 62/459,920,which were filed on Feb. 16, 2017, all of which applications areincorporated herein in their entireties for all purposes.

In some embodiments, the adaptor comprises a receptacle slot, which isconfigured to accommodate the QMAX device when the device is in a closedconfiguration. In certain embodiments, the QMAX device has a sampledeposited therein and the adaptor can be connected to a mobile device(e.g. a smartphone) so that the sample can be read by the mobile device.In certain embodiments, the mobile device can detect and/or analyze asignal from the sample. In certain embodiments, the mobile device cancapture images of the sample when the sample is in the QMAX device andpositioned in the field of view (FOV) of a camera, which in certainembodiments, is part of the mobile device.

In some embodiments, the adaptor comprises optical components, which areconfigured to enhance, magnify, and/or optimize the production of thesignal from the sample. In some embodiments, the optical componentsinclude parts that are configured to enhance, magnify, and/or optimizeillumination provided to the sample. In certain embodiments, theillumination is provided by a light source that is part of the mobiledevice. In some embodiments, the optical components include parts thatare configured to enhance, magnify, and/or optimize a signal from thesample.

(6) Smartphone Detection System

The devices/apparatus, systems, and methods herein disclosed can includeor use Q-cards for sample detection, analysis, and quantification. Insome embodiments, the Q-card is used together with an adaptor that canconnect the Q-card with a smartphone detection system. In someembodiments, the smartphone comprises a camera and/or an illuminationsource The smartphone detection system, as well the associated hardwareand software are herein disclosed, listed, described, and/or summarizedin PCT Application (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application Nos. 62/456,287 and62/456,590, which were filed on Feb. 8, 2017, U.S. ProvisionalApplication No. 62/456,504, which was filed on Feb. 8, 2017, U.S.Provisional Application No. 62/459,544, which was filed on Feb. 15,2017, and U.S. Provisional Application No. 62/460,075 and 62/459,920,which were filed on Feb. 16, 2017, all of which applications areincorporated herein in their entireties for all purposes.

In some embodiments, the smartphone comprises a camera, which can beused to capture images or the sample when the sample is positioned inthe field of view of the camera (e.g. by an adaptor). In certainembodiments, the camera includes one set of lenses (e.g. as in iPhone™6). In certain embodiments, the camera includes at least two sets oflenses (e.g. as in iPhone™ 7). In some embodiments, the smartphonecomprises a camera, but the camera is not used for image capturing.

In some embodiments, the smartphone comprises a light source such as butnot limited to LED (light emitting diode). In certain embodiments, thelight source is used to provide illumination to the sample when thesample is positioned in the field of view of the camera (e.g. by anadaptor). In some embodiments, the light from the light source isenhanced, magnified, altered, and/or optimized by optical components ofthe adaptor.

In some embodiments, the smartphone comprises a processor that isconfigured to process the information from the sample. The smartphoneincludes software instructions that, when executed by the processor, canenhance, magnify, and/or optimize the signals (e.g. images) from thesample. The processor can include one or more hardware components, suchas a central processing unit (CPU), an application-specific integratedcircuit (ASIC), an application-specific instruction-set processor(ASIP), a graphics processing unit (GPU), a physics processing unit(PPU), a digital signal processor (DSP), a field-programmable gate array(FPGA), a programmable logic device (PLD), a controller, amicrocontroller unit, a reduced instruction-set computer (RISC), amicroprocessor, or the like, or any combination thereof.

In some embodiments, the smartphone comprises a communication unit,which is configured and/or used to transmit data and/or images relatedto the sample to another device. Merely by way of example, thecommunication unit can use a cable network, a wireline network, anoptical fiber network, a telecommunications network, an intranet, theInternet, a local area network (LAN), a wide area network (WAN), awireless local area network (WLAN), a metropolitan area network (MAN), awide area network (WAN), a public telephone switched network (PSTN), aBluetooth network, a ZigBee network, a near field communication (NFC)network, or the like, or any combination thereof.

In some embodiments, the smartphone is an iPhone™, an Android™ phone, ora Windows™ phone.

(7) Detection Methods

The devices/apparatus, systems, and methods herein disclosed can includeor be used in various types of detection methods. The detection methodsare herein disclosed, listed, described, and/or summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application Nos. 62/456,287,62/456,528, 62/456,631, 62/456,522, 62/456,598, 62/456,603, and62/456,628, which were filed on Feb. 8, 2017, U.S. ProvisionalApplication No. 62/459,276, 62/456,904, 62/457,075, and 62/457,009,which were filed on Feb. 9, 2017, and U.S. Provisional Application No.62/459,303, 62/459,337, and 62/459,598, which were filed on Feb. 15,2017, and U.S. Provisional Application No. 62/460,083, 62/460,076, whichwere filed on Feb. 16, 2017, all of which applications are incorporatedherein in their entireties for all purposes.

(8) Labels, Capture Agent and Detection Agent

The devices/apparatus, systems, and methods herein disclosed can employvarious types of labels, capture agents, and detection agents that areused for analytes detection. The labels are herein disclosed, listed,described, and/or summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional Application No.62/456,065, which was filed on Feb. 7, 2017, U.S. ProvisionalApplication No. 62/456,287, which was filed on Feb. 8, 2017, and U.S.Provisional Application No. 62/456,504, which was filed on Feb. 8, 2017,all of which applications are incorporated herein in their entiretiesfor all purposes.

In some embodiments, the label is optically detectable, such as but notlimited to a fluorescence label. In some embodiments, the labelsinclude, but are not limited to, IRDye800CW, Alexa 790, Dylight 800,fluorescein, fluorescein isothiocyanate, succinimidyl esters ofcarboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer offluorescein dichlorotriazine, cagedcarboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine,Texas Red, propidium iodide, JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanineiodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethylrhodamine methyl ester), TMRE (tetramethyl rhodamine ethyl ester),tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine,green fluorescent protein, blue-shifted green fluorescent protein,cyan-shifted green fluorescent protein, red-shifted green fluorescentprotein, yellow-shifted green fluorescent protein,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives, such as acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-cacid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives:coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriaamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2-,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-(dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)amino- -fluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelli-feroneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl hodamine isothiocyanate (TRITC); riboflavin;5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CALFluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7;IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine,coumarins and related dyes, xanthene dyes such as rhodols, resorufins,bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazidessuch as luminol, and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, fluorescent europium and terbium complexes;combinations thereof, and the like. Suitable fluorescent proteins andchromogenic proteins include, but are not limited to, a greenfluorescent protein (GFP), including, but not limited to, a GFP derivedfrom Aequoria victoria or a derivative thereof, e.g., a “humanized”derivative such as Enhanced GFP; a GFP from another species such asRenilla reniformis, Renilla mulleri, or Ptilosarcus guernyi; “humanized”recombinant GFP (hrGFP); any of a variety of fluorescent and coloredproteins from Anthozoan species; combinations thereof; and the like.

In any embodiment, the QMAX device can contain a plurality of captureagents and/or detection agents that each bind to a biomarker selectedfrom Tables B1, B2, B3 and/or B7 in U.S. Provisional Application No.62/234,538 and/or PCT Application No. PCT/US2016/054025, wherein thereading step d) includes obtaining a measure of the amount of theplurality of biomarkers in the sample, and wherein the amount of theplurality of biomarkers in the sample is diagnostic of a disease orcondition.

In any embodiment, the capture agent and/or detection agents can be anantibody epitope and the biomarker can be an antibody that binds to theantibody epitope. In some embodiments, the antibody epitope includes abiomolecule, or a fragment thereof, selected from Tables B4, B5 or B6 inU.S. Provisional Application No. 62/234,538 and/or PCT Application No.PCT/US2016/054025. In some embodiments, the antibody epitope includes anallergen, or a fragment thereof, selected from Table B5. In someembodiments, the antibody epitope includes an infectious agent-derivedbiomolecule, or a fragment thereof, selected from Table B6 in U.S.Provisional Application No. 62/234,538 and/or PCT Application No.PCT/US2016/054025.

In any embodiment, the QMAX device can contain a plurality of antibodyepitopes selected from Tables B4, B5 and/or B6 in U.S. ProvisionalApplication No. 62/234,538 and/or PCT Application No. PCT/US2016/054025,wherein the reading step d) includes obtaining a measure of the amountof a plurality of epitope-binding antibodies in the sample, and whereinthe amount of the plurality of epitope-binding antibodies in the sampleis diagnostic of a disease or condition.

(9) Analytes

The devices/apparatus, systems, and methods herein disclosed can beapplied to manipulation and detection of various types of analytes(including biomarkers). The analytes are herein disclosed, listed,described, and/or summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional Application No.62/456,065, which was filed on Feb. 7, 2017, U.S. ProvisionalApplication No. 62/456,287, which was filed on Feb. 8, 2017, and U.S.Provisional Application No. 62/456,504, which was filed on Feb. 8, 2017,all of which applications are incorporated herein in their entiretiesfor all purposes.

The devices, apparatus, systems, and methods herein disclosed can beused for the detection, purification and/or quantification of variousanalytes. In some embodiments, the analytes are biomarkers thatassociated with various diseases. In some embodiments, the analytesand/or biomarkers are indicative of the presence, severity, and/or stageof the diseases. The analytes, biomarkers, and/or diseases that can bedetected and/or measured with the devices, apparatus, systems, and/ormethod of the present invention include the analytes, biomarkers, and/ordiseases listed, described and/or summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 filed on Aug. 10, 2016, andPCT Application No. PCT/US2016/054025 filed on Sep. 27, 2016, and U.S.Provisional Application Nos. 62/234,538 filed on Sep. 29, 2015,62/233,885 filed on Sep. 28, 2015, 62/293,188 filed on Feb. 9, 2016, and62/305,123 filed on Mar. 8, 2016, which are all hereby incorporated byreference by their entireties. For example, the devices, apparatus,systems, and methods herein disclosed can be used in (a) the detection,purification and quantification of chemical compounds or biomoleculesthat correlates with the stage of certain diseases, e.g., infectious andparasitic disease, injuries, cardiovascular disease, cancer, mentaldisorders, neuropsychiatric disorders and organic diseases, e.g.,pulmonary diseases, renal diseases, (b) the detection, purification andquantification of microorganism, e.g., virus, fungus and bacteria fromenvironment, e.g., water, soil, or biological samples, e.g., tissues,bodily fluids, (c) the detection, quantification of chemical compoundsor biological samples that pose hazard to food safety or nationalsecurity, e.g. toxic waste, anthrax, (d) quantification of vitalparameters in medical or physiological monitor, e.g., glucose, bloodoxygen level, total blood count, (e) the detection and quantification ofspecific DNA or RNA from biosamples, e.g., cells, viruses, bodilyfluids, (f) the sequencing and comparing of genetic sequences in DNA inthe chromosomes and mitochondria for genome analysis or (g) to detectreaction products, e.g., during synthesis or purification ofpharmaceuticals.

In some embodiments, the analyte can be a biomarker, an environmentalmarker, or a foodstuff marker. The sample in some instances is a liquidsample, and can be a diagnostic sample (such as saliva, serum, blood,sputum, urine, sweat, lacrima, semen, or mucus); an environmental sampleobtained from a river, ocean, lake, rain, snow, sewage, sewageprocessing runoff, agricultural runoff, industrial runoff, tap water ordrinking water; or a foodstuff sample obtained from tap water, drinkingwater, prepared food, processed food or raw food.

In any embodiment, the sample can be a diagnostic sample obtained from asubject, the analyte can be a biomarker, and the measured the amount ofthe analyte in the sample can be diagnostic of a disease or a condition.

In any embodiment, the devices, apparatus, systems, and methods in thepresent invention can further include diagnosing the subject based oninformation including the measured amount of the biomarker in thesample. In some cases, the diagnosing step includes sending datacontaining the measured amount of the biomarker to a remote location andreceiving a diagnosis based on information including the measurementfrom the remote location.

In any embodiment, the biomarker can be selected from Tables B1, 2, 3 or7 as disclosed in U.S. Provisional Application Nos. 62/234,538,62/293,188, and/or 62/305,123, and/or PCT Application No.PCT/US2016/054,025, which are all incorporated in their entireties forall purposes. In some instances, the biomarker is a protein selectedfrom Tables B1, 2, or 3. In some instances, the biomarker is a nucleicacid selected from Tables B2, 3 or 7. In some instances, the biomarkeris an infectious agent-derived biomarker selected from Table B2. In someinstances, the biomarker is a microRNA (miRNA) selected from Table B7.

In any embodiment, the applying step b) can include isolating miRNA fromthe sample to generate an isolated miRNA sample, and applying theisolated miRNA sample to the disk-coupled dots-on-pillar antenna (QMAXdevice) array.

In any embodiment, the QMAX device can contain a plurality of captureagents that each bind to a biomarker selected from Tables B1, B2, B3and/or B7, wherein the reading step d) includes obtaining a measure ofthe amount of the plurality of biomarkers in the sample, and wherein theamount of the plurality of biomarkers in the sample is diagnostic of adisease or condition.

In any embodiment, the capture agent can be an antibody epitope and thebiomarker can be an antibody that binds to the antibody epitope. In someembodiments, the antibody epitope includes a biomolecule, or a fragmentthereof, selected from Tables B4, B5 or B6. In some embodiments, theantibody epitope includes an allergen, or a fragment thereof, selectedfrom Table B5. In some embodiments, the antibody epitope includes aninfectious agent-derived biomolecule, or a fragment thereof, selectedfrom Table B6.

In any embodiment, the QMAX device can contain a plurality of antibodyepitopes selected from Tables B4, B5 and/or B6, wherein the reading stepd) includes obtaining a measure of the amount of a plurality ofepitope-binding antibodies in the sample, and wherein the amount of theplurality of epitope-binding antibodies in the sample is diagnostic of adisease or condition.

In any embodiment, the sample can be an environmental sample, andwherein the analyte can be an environmental marker. In some embodiments,the environmental marker is selected from Table B8 in U.S. ProvisionalApplication No. 62/234,538 and/or PCT Application No. PCT/US2016/054025.

In any embodiment, the method can include receiving or providing areport that indicates the safety or harmfulness for a subject to beexposed to the environment from which the sample was obtained.

In any embodiment, the method can include sending data containing themeasured amount of the environmental marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to be exposed to the environment from which the sample wasobtained.

In any embodiment, the QMAX device array can include a plurality ofcapture agents that each binds to an environmental marker selected fromTable B8, and wherein the reading step d) can include obtaining ameasure of the amount of the plurality of environmental markers in thesample.

In any embodiment, the sample can be a foodstuff sample, wherein theanalyte can be a foodstuff marker, and wherein the amount of thefoodstuff marker in the sample can correlate with safety of thefoodstuff for consumption. In some embodiments, the foodstuff marker isselected from Table B9.

In any embodiment, the method can include receiving or providing areport that indicates the safety or harmfulness for a subject to consumethe foodstuff from which the sample is obtained.

In any embodiment, the method can include sending data containing themeasured amount of the foodstuff marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to consume the foodstuff from which the sample is obtained.

In any embodiment, the devices, apparatus, systems, and methods hereindisclosed can include a plurality of capture agents that each binds to afoodstuff marker selected from Table B9 from in U.S. ProvisionalApplication No. 62/234,538 and PCT Application No. PCT/US2016/054025,wherein the obtaining can include obtaining a measure of the amount ofthe plurality of foodstuff markers in the sample, and wherein the amountof the plurality of foodstuff marker in the sample can correlate withsafety of the foodstuff for consumption.

Also provided herein are kits that find use in practicing the devices,systems and methods in the present invention.

The amount of sample can be about a drop of a sample. The amount ofsample can be the amount collected from a pricked finger or fingerstick.The amount of sample can be the amount collected from a microneedle or avenous draw.

A sample can be used without further processing after obtaining it fromthe source, or can be processed, e.g., to enrich for an analyte ofinterest, remove large particulate matter, dissolve or resuspend a solidsample, etc.

Any suitable method of applying a sample to the QMAX device can beemployed. Suitable methods can include using a pipet, dropper, syringe,etc. In certain embodiments, when the QMAX device is located on asupport in a dipstick format, as described below, the sample can beapplied to the QMAX device by dipping a sample-receiving area of thedipstick into the sample.

A sample can be collected at one time, or at a plurality of times.Samples collected over time can be aggregated and/or processed (byapplying to a QMAX device and obtaining a measurement of the amount ofanalyte in the sample, as described herein) individually. In someinstances, measurements obtained over time can be aggregated and can beuseful for longitudinal analysis over time to facilitate screening,diagnosis, treatment, and/or disease prevention.

Washing the QMAX device to remove unbound sample components can be donein any convenient manner, as described above. In certain embodiments,the surface of the QMAX device is washed using binding buffer to removeunbound sample components.

Detectable labeling of the analyte can be done by any convenient method.The analyte can be labeled directly or indirectly. In direct labeling,the analyte in the sample is labeled before the sample is applied to theQMAX device. In indirect labeling, an unlabeled analyte in a sample islabeled after the sample is applied to the QMAX device to capture theunlabeled analyte, as described below.

(10) Applications

The devices/apparatus, systems, and methods herein disclosed can be usedfor various applications (fields and samples). The applications areherein disclosed, listed, described, and/or summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

In some embodiments, the devices, apparatus, systems, and methods hereindisclosed are used in a variety of different application in variousfield, wherein determination of the presence or absence, quantification,and/or amplification of one or more analytes in a sample are desired.For example, in certain embodiments the subject devices, apparatus,systems, and methods are used in the detection of proteins, peptides,nucleic acids, synthetic compounds, inorganic compounds, organiccompounds, bacteria, virus, cells, tissues, nanoparticles, and othermolecules, compounds, mixtures and substances thereof. The variousfields in which the subject devices, apparatus, systems, and methods canbe used include, but are not limited to: diagnostics, management, and/orprevention of human diseases and conditions, diagnostics, management,and/or prevention of veterinary diseases and conditions, diagnostics,management, and/or prevention of plant diseases and conditions,agricultural uses, veterinary uses, food testing, environments testingand decontamination, drug testing and prevention, and others.

The applications of the present invention include, but are not limitedto: (a) the detection, purification, quantification, and/oramplification of chemical compounds or biomolecules that correlates withcertain diseases, or certain stages of the diseases, e.g., infectiousand parasitic disease, injuries, cardiovascular disease, cancer, mentaldisorders, neuropsychiatric disorders and organic diseases, e.g.,pulmonary diseases, renal diseases, (b) the detection, purification,quantification, and/or amplification of cells and/or microorganism,e.g., virus, fungus and bacteria from the environment, e.g., water,soil, or biological samples, e.g., tissues, bodily fluids, (c) thedetection, quantification of chemical compounds or biological samplesthat pose hazard to food safety, human health, or national security,e.g. toxic waste, anthrax, (d) the detection and quantification of vitalparameters in medical or physiological monitor, e.g., glucose, bloodoxygen level, total blood count, (e) the detection and quantification ofspecific DNA or RNA from biological samples, e.g., cells, viruses,bodily fluids, (f) the sequencing and comparing of genetic sequences inDNA in the chromosomes and mitochondria for genome analysis or (g) thedetection and quantification of reaction products, e.g., duringsynthesis or purification of pharmaceuticals.

In some embodiments, the subject devices, apparatus, systems, andmethods are used in the detection of nucleic acids, proteins, or othermolecules or compounds in a sample. In certain embodiments, the devices,apparatus, systems, and methods are used in the rapid, clinicaldetection and/or quantification of one or more, two or more, or three ormore disease biomarkers in a biological sample, e.g., as being employedin the diagnosis, prevention, and/or management of a disease conditionin a subject. In certain embodiments, the devices, apparatus, systems,and methods are used in the detection and/or quantification of one ormore, two or more, or three or more environmental markers in anenvironmental sample, e.g. sample obtained from a river, ocean, lake,rain, snow, sewage, sewage processing runoff, agricultural runoff,industrial runoff, tap water or drinking water. In certain embodiments,the devices, apparatus, systems, and methods are used in the detectionand/or quantification of one or more, two or more, or three or morefoodstuff marks from a food sample obtained from tap water, drinkingwater, prepared food, processed food or raw food.

In some embodiments, the subject device is part of a microfluidicdevice. In some embodiments, the subject devices, apparatus, systems,and methods are used to detect a fluorescence or luminescence signal. Insome embodiments, the subject devices, apparatus, systems, and methodsinclude, or are used together with, a communication device, such as butnot limited to: mobile phones, tablet computers and laptop computers. Insome embodiments, the subject devices, apparatus, systems, and methodsinclude, or are used together with, an identifier, such as but notlimited to an optical barcode, a radio frequency ID tag, or combinationsthereof.

In some embodiments, the sample is a diagnostic sample obtained from asubject, the analyte is a biomarker, and the measured amount of theanalyte in the sample is diagnostic of a disease or a condition. In someembodiments, the subject devices, systems and methods further includereceiving or providing to the subject a report that indicates themeasured amount of the biomarker and a range of measured values for thebiomarker in an individual free of or at low risk of having the diseaseor condition, wherein the measured amount of the biomarker relative tothe range of measured values is diagnostic of a disease or condition.

In some embodiments, the sample is an environmental sample, and whereinthe analyte is an environmental marker. In some embodiments, the subjectdevices, systems and methods includes receiving or providing a reportthat indicates the safety or harmfulness for a subject to be exposed tothe environment from which the sample was obtained. In some embodiments,the subject devices, systems and methods include sending data containingthe measured amount of the environmental marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to be exposed to the environment from which the sample wasobtained.

In some embodiments, the sample is a foodstuff sample, wherein theanalyte is a foodstuff marker, and wherein the amount of the foodstuffmarker in the sample correlate with safety of the foodstuff forconsumption. In some embodiments, the subject devices, systems andmethods include receiving or providing a report that indicates thesafety or harmfulness for a subject to consume the foodstuff from whichthe sample is obtained. In some embodiments, the subject devices,systems and methods include sending data containing the measured amountof the foodstuff marker to a remote location and receiving a report thatindicates the safety or harmfulness for a subject to consume thefoodstuff from which the sample is obtained.

(11) Dimensions

The devices, apparatus, systems, and methods herein disclosed caninclude or use a QMAX device, which can comprise plates and spacers. Insome embodiments, the dimension of the individual components of the QMAXdevice and its adaptor are listed, described and/or summarized in PCTApplication (designating U.S.) No. PCT/US2016/045437 filed on Aug. 10,2016, and U.S. Provisional Application Nos. 62/431,639 filed on Dec. 9,2016 and 62/456,287 filed on Feb. 8, 2017, which are all herebyincorporated by reference by their entireties.

(12) Cloud

The devices/apparatus, systems, and methods herein disclosed can employcloud technology for data transfer, storage, and/or analysis. Therelated cloud technologies are herein disclosed, listed, described,and/or summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional Application No.62/456,065, which was filed on Feb. 7, 2017, U.S. ProvisionalApplication No. 62/456,287, which was filed on Feb. 8, 2017, and U.S.Provisional Application No. 62/456,504, which was filed on Feb. 8, 2017,all of which applications are incorporated herein in their entiretiesfor all purposes.

In some embodiments, the cloud storage and computing technologies caninvolve a cloud database. Merely by way of example, the cloud platformcan include a private cloud, a public cloud, a hybrid cloud, a communitycloud, a distributed cloud, an inter-cloud, a multi-cloud, or the like,or any combination thereof. In some embodiments, the mobile device (e.g.smartphone) can be connected to the cloud through any type of network,including a local area network (LAN) or a wide area network (WAN).

In some embodiments, the data (e.g. images of the sample) related to thesample is sent to the cloud without processing by the mobile device andfurther analysis can be conducted remotely. In some embodiments, thedata related to the sample is processed by the mobile device and theresults are sent to the cloud. In some embodiments, both the raw dataand the results are transmitted to the cloud.

What is claimed is:
 1. A device for analyzing white blood cells in ablood sample, comprising: a first plate, a second plate, and spacers,wherein: vi. the plates are movable relative to each other intodifferent configurations; vii. one or both plates are flexible; viii.each of the plates comprises an inner surface that has a sample contactarea for contacting a blood sample; ix. one or both of the platescomprise the spacers that are permanently fixed on the sample contactarea of a respective plate; x. the spacers have: (a) a predeterminedsubstantially uniform height that has a value selected in the range of 5um to 50 um, (b) a shape of pillar with substantially uniformcross-section and a flat top surface; (c) a ratio of the width to theheight equal to or larger than one; (g) a predetermined, fixed,non-random, inter-spacer distance that is in the range of 10 um to 200um (micron); (e) a filling factor of equal to 1% or larger, wherein thefilling factor is the ratio of the spacer contact area (on the plate) tothe total plate area; and (f) the filling factor multiplies the Young'smodulus of the spacer is equal to 2 MPa or larger; wherein one of theconfigurations is an open configuration, in which: the two plates arepartially or completely separated apart, the spacing between the platesis not regulated by the spacers, and the sample is deposited on one orboth of the plates; wherein another of the configurations is a closedconfiguration which is configured after the sample is deposited in theopen configuration; and in the closed configuration: at least part ofthe sample is compressed by the two plates into a layer of highlyuniform thickness and is substantially stagnant relative to the plates,wherein the uniform thickness of the layer is confined by the samplecontact areas of the two plates and is regulated by the plates and thespacers.
 2. The device of claim 1, wherein the analyte is white bloodcells (WBC).
 3. The device of claim 1, wherein the gap size of device isin the range of 2 um to 150 um.
 4. The device of claim 1, wherein thepreferred gap size of device is in the range of 5 um to 50 um.
 5. Thedevice of claim 1, wherein the preferred gap size of device is in therange of 10 um to 30 um.
 6. The device of claim 1, wherein the preferredgap size of device is 30 um.
 7. The device of claim 1, wherein wherein apreferred field of view for counting and differentiating WBCs is atleast 0.1 mm², 10 mm², 50 mm², 100 mm² or a range between any two of thevalues.
 8. The device of claim 1, wherein wherein a preferred field ofview for counting and differentiating WBCs is in the range of 1 mm² to50 mm².
 9. The device of claim 1, wherein wherein a preferred field ofview for counting and differentiating WBCs is in the range of 10 mm² to30 mm².
 10. The device of claim 1, wherein wherein a preferred field ofview for counting and differentiating WBCs is in the range of 20 mm².11. The device of claim 1, wherein the field of view is in the range of10 mm² to 50 mm², the preferred gap size of device is in the range of 10um to 50 um, thereby the WBC counting and WBC differentiation precisionand accuracy is less than 15%.
 12. The device of claim 1, wherein thefield of view is in the range of 10 mm² to 30 mm², the preferred gapsize of device is in the range of 20 um to 30 um, thereby the WBCcounting and WBC differentiation precision and accuracy is less than15%.
 13. The device of claim 1, wherein the field of view is in therange of 1 mm² to 10 mm², the preferred gap size of device is in therange of 50 um to 150 um, thereby the WBC counting and WBCdifferentiation precision and accuracy is less than 15%.
 14. The deviceof claim 1, wherein the anti-conglutination agent is coated inside thedevice, comprises ethylenediaminetetraacetic acid (EDTA), EDTA disodium,K₂EDTA, or K₃EDTA, citrate, heparin or any combinations thereof.
 15. Thedevice of claim 1, wherein the analyte is marked with fluorescencereagents.
 16. The device of claim 1, wherein the analyte is marked withcolorimetric reagents.
 17. The device of claim 1, wherein the cell stainagent is coated inside the device, comprises Wright's stain (Eosin,methylene blue), Giemsa stain (Eosin, methylene blue, and Azure B),May-Grünwald stain, Leishman's stain (“Polychromed” methylene blue (i.e.demethylated into various azures) and eosin), Erythrosine B stain(Erythrosin B), and other fluorescence stain including but not limit toAcridine orange dye, 3,3-dihexyloxacarbocyanine (DiOC6), PropidiumIodide (PI), Fluorescein Isothiocyanate (FITC) and Basic Orange 21(BO21) dye, Ethidium Bromide, Brilliant Sulfaflavine and a StilbeneDisulfonic Acid derivative, Erythrosine B or trypan blue, Hoechst 33342,Trihydrochloride, Trihydrate, or DAPI (4′,6-Diamidino-2-Phenylindole,Dihydrochloride), YOYO or any combinations thereof.
 18. The device ofclaim 1, wherein the cell lysing agent is coated inside the device,comprises ammonium chloride, sodium bicarbonate,ethylenediaminetetraacetic acid (EDTA), acetic acid, citric acid, orother acid and base, or any combinations thereof.
 19. The device ofclaim 1, wherein the cell distribution and lysing agent is coated insidethe device, comprises but not limit to Zwittergent, ASB-14, ASB-16,CHAPS, Cationic surfactant NN-[Tris(hydroxymethyl)methyl]-N-alkyl-N,N-dimethyl ammonium chloride (IIa), IIb, IIc, IId,CTAC, Tween 20, Tween 40, Tween 60, Tween 80, Sodium lauryl sulfate(SLS), ammonium lauryl sulfate, CTAB, sodium lauryl ether sulfate(SLES), sodium myreth sulfate, docusate, perfluorooctanesulfonate,alkyl-aryl ether phosphates, alkyl ether phosphates, CTAB,cetylpyridinium chloride (CPC), benzalkonium chloride (BAC),benzethonium chloride (BZT), dimethyldioctadecylammonium chloride,dioctadecyldimethlyammonium bromide (DODAB), cocamidopropylhydroxysultaine, cocamidopropyl betaine, narrow-range ethoxylate,octaethylene glycol monododecyl ether, pentaethylene glycol monododecylether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamidemonoethanolamine, cocamide diethanolamine, poloxamers, glycerolmonostearate, glycerol monolaurate, sorbitan monolaurate, sorbitanmonostearate, sorbitan tristearate, decyl glucoside, lauryl glucoside,octyl glucoside, lauryldimethylamine oxide, dimethyl sulfoxide,phosphine oxide.
 20. The device of claim 1, wherein the celldistribution and lysing agent is coated inside the device, comprisesPluronic F-127, Cremophor EL, Pluronic F-68, Myrj 52, Brij 35, sodiumoleate, sodium dodecyl sulfate, Tween 20, Tween 40, Tween 60, Tween 80,SLS, CTAB, CTAC, Tamoxifen, saponin, hydrochloric acid, sulfuric acid,nitric acid, phosphoric acid, lactic acid, ABS-14, ABS-16, anti-malariadrugs (quinine compounds), arsenic, dapsone, metals (chromium/chromates,platinum salts, nickel compounds, copper, lead, cis-platinum), nitrites,nitrofurantoin, penicillin, phenazopyridine (pyridium), rho immuneglobulin, ribavirin, sulfonamides, sulfones.
 21. The device of claim 1,wherein the release time control material is coated inside the device,comprises albumin, carbomers, carboxymethyl cellulose, carrageenan,chitosan, dextrin, polyethylene glycol, polyvinylpyrrolidone, orpolyvinyl alcohol, or any combinations thereof.
 22. An adapter devicefor analyzing an analyte in a liquid sample, comprising: (k) anattachment member configured to attach the adapter to an apparatus thatcomprises a light source and a camera; (l) a card slot configured toaccommodate a sample card, which contains a liquid sample that iscompressed into a layer of uniform thickness, wherein when the samplecard inserted into the card slot, the sample is positioned under theview of the camera and the light source; (m) an optical filterconfigured to filter light from the light source to form a first beamwith a specific wavelength range, wherein a part of the first beamilluminates on the edge of the sample card and travels in the samplecard to illuminate the sample; (n) a mirror configured to deflect partof the first beam to form a second beam that back-illuminates the samplein an oblique angle; (o) an absorber configured to absorb a remainingpart of the first beam that has a divergence angle.
 23. The adaptor ofclaim 22, wherein the lens is positioned on a front-side of the sampleand the mirror is positioned to obliquely illuminate the sample from aback-side of the sample, wherein the oblique angle is larger than acollecting angle of the lens.
 24. The adaptor of claim 22, furthercomprising a wavelength filter positioned between the sample and thecamera to pass fluorescence emitted by the sample in response to theoblique illumination.
 25. The adaptor of claim 22, wherein the samplecard is supported by a sample holder comprising a planar structure, andwherein the receptacle sample slot is configured to position the planarstructure to extend partially into a path of illumination light from thelight source to couple illumination light into the planar structure. 26.A method for analyzing an analyte in a liquid sample, comprising: (a)obtaining the liquid sample; (b) compressing at least part of the sampleinto a layer of uniform thickness with a sample card, (c) inserting thesample card into an adaptor device, which is configured to be attachedto an apparatus that comprises a light source and a camera; (d)illuminating the sample with light from the light source, wherein i. thelight is filtered by an optical filter of the adapter device to form afirst beam with a specific wavelength range, part of the first beamilluminating on the edge of the sample card and travels in the samplecard to illuminate the sample; ii. part of the first beam is deflectedby a mirror of the adapter device to form a second beam thatback-illuminates the sample in an oblique angle; and iii. a remainingpart of the first beam that has a divergence angle is absorbed by anabsorber of the adapter device.
 27. The method of claim 26, furthercomprising: (e) capturing images of the sample in the layer of uniformthickness with the camera; (f) analyzing the images to enumerate theanalyte in the images; and (g) calculating the concentration of theanalyte in the sample based on the uniform thickness, a field of view ofthe camera, analyte number, and a predetermined correction factor;wherein the field of view is the extent of the field in which the cameracaptures the images; wherein the correction factor is determined by amiscount ratio, which is dependent on the field of view, the uniformthickness, and properties of the analyte.
 28. A method for analyzingwhite blood cells in a blood sample, comprising: (a) obtaining a bloodsample; (b) obtaining a device of claim 1; (c) depositing the bloodsample on one or both of the plates when the plates are configured inthe open configuration, (d) after (c), forcing the two plates into aclosed configuration; and (e) capturing images of sample in the layer ofuniform thickness while the plates are the closed configuration; and (h)analyzing the images to determine the concentration of white blood cellsin the sample.
 29. A method for white blood cell and sub-type (includingneutrophils, eosinophils, basophils, lymphocytes, and monocytes)counting using a single device, comprising: (a) obtaining a bloodsample; (b) obtaining the device of any prior claims, wherein the spacerheight is 5 um to 50 um, (c) depositing the blood sample on one or bothof the plates when the plates are configured in an open configuration;(d) after (c), forcing the two plates into a closed configuration; (e)capturing images of the sample in the layer of uniform thickness whilethe plates are the closed configuration; and (f) analyzing the images todetermine the respective number of white blood cells, neutrophils,lymphocytes, monocytes, eosinophils and basophils, through the countingof the cell number in the image and the analysis of the fluorescencecolor and shape for each white blood cell.
 30. A method for analyzing ananalyte in a liquid sample, comprising: (a) obtaining the liquid sample;(b) compressing at least part of the sample into a layer of uniformthickness, (c) capturing images of the sample in the layer of uniformthickness with a camera, wherein the images show the analyte; and (d)analyzing the images to enumerate the analyte in the images, (e)calculating the concentration of the analyte in the sample based on theuniform thickness, a field of view of the camera, the analyteenumeration, and a predetermined correction factor; wherein the field ofview is the extent of the field in which the camera captures the images;wherein the correction factor is determined by a miscount ratio, whichis dependent on the field of view, the uniform thickness, and propertiesof the analyte.
 31. The method of claim 27, wherein the correctionfactor is used to back calculate the WBC count to WBC concentration tocompensate the WBC miss counting in the process.
 32. The method of claim27, wherein the correction factor depending on the gap size of device isused to back calculate the WBC count to WBC concentration to compensatethe WBC miss counting in the process.
 33. The method of claim 27,wherein the correction factor is used to back calculate the WBC count toWBC concentration to compensate other imperfection effect as WBCditribution in the process.